JP6963667B2 - Encapsulation of messenger RNA - Google Patents

Description

Cross-references of related applications This application is incorporated herein by reference in its entirety, 2014.
Claims priority over US Provisional Patent Application No. 62 / 020,163, filed July 2, 2014.

Messenger RNA therapy (MRT) has become an increasingly important technique for the treatment of various diseases. MRT necessarily involves administration of messenger RNA (mRNA) to a patient who needs treatment for the production of a protein encoded by mRNA in the patient's body. Lipid nanoparticles are often used to encapsulate mRNA for efficient in vivo delivery of mRNA. However, current methods for producing mRNA-carrying lipid nanoparticles suffer from poor encapsulation efficiency, low mRNA recovery and / or heterogeneous particle size.

The present invention provides, among other things, improved processes for lipid nanoparticle formulations and mRNA encapsulation. In particular, in the present invention, preheating the mRNA solution and / or lipid solution prior to mixing significantly improved encapsulation efficiency, mRNA recovery, and more homogeneous and smaller particle size (eg, less than 100 nm). ) Is based on the surprising discovery that it brings.

Therefore, in some embodiments, the present invention is the process of encapsulating messenger RNA (mRNA) in lipid nanoparticles, comprising mixing the mRNA solution with the lipid solution, comprising the steps of mixing the mRNA solution and / or the lipid. The process is provided, wherein the solution is at a predetermined temperature above the ambient temperature. In some embodiments, a predetermined temperature suitable for the present invention is
It is about 30 ° C., 37 ° C., 40 ° C., 45 ° C., 50 ° C., 55 ° C., 60 ° C., 65 ° C., or 70 ° C. or higher. In some embodiments, the predetermined temperatures suitable for the present invention are about 25-70 ° C, about 30-70 ° C, about 35-70 ° C, about 40-70 ° C, about 45-70 ° C, about 50-. It is in the range of 70 ° C., or about 60-70 ° C. In certain embodiments, a predetermined temperature suitable for the present invention is
It is about 65 ° C.

In some embodiments, the mRNA and lipid solutions are separately heated to a predetermined temperature prior to mixing. In some embodiments, the mRNA solution is heated to a predetermined temperature and the lipid solution is at ambient temperature prior to mixing. In some embodiments, the ambient temperature mRN
The mRNA solution is heated to a predetermined temperature by adding the A stock solution to the heated buffer solution to a predetermined temperature. In some embodiments, the buffer has a pH of about 4.5 or less (eg, about 4.4, 4.2, 4.0 or 3.8 or less).

In some embodiments, the mRNA and lipid solutions are mixed by a low pulse flow pump. In some embodiments, a suitable pump is a gear pump. In some embodiments, a suitable pump is a peristaltic pump. In some embodiments, a suitable pump is a centrifugal pump.

In some embodiments, the mRNA solution is about 150-250 ml / min, 250-50.
0 ml / min, 500-1000 ml / min, 1000-2000 ml / min, 2000-30
Mix at a flow rate in the range of 00 ml / min, 3000-4000 ml / min, or 4000-5000 ml / min. In some embodiments, the mRNA solution is about 200 ml / min, about 500 ml / min, about 1000 ml / min, about 2000 ml / min, about 3000 ml / min, about 4
Mix at a flow rate of 000 ml / min, or about 5000 ml / min.

In some embodiments, the lipid solution is about 25-75 ml / min, about 75-200 ml / min.
Mix at flow rates in the range of min, about 200-350 ml / min, about 350-500 ml / min, about 500-650 ml / min, about 650-850 ml / min, or about 850-1000 ml / min. In some embodiments, the lipid solution is about 50 ml / min, about 100 ml / min, about 1
50 ml / min, about 200 ml / min, about 250 ml / min, about 300 ml / min, about 350 ml
/ Min, about 400 ml / min, about 450 ml / min, about 500 ml / min, about 550 ml / min, about 600 ml / min, about 650 ml / min, about 700 ml / min, about 750 ml / min, about 800 m
l / min, about 850 ml / min, about 900 ml / min, about 950 ml / min, or about 1000 m
Mix at a flow rate of l / min.

In some embodiments, the process according to the invention comprises first producing an mRNA solution by mixing the citrate buffer with the mRNA stock solution. In certain embodiments, a suitable citrate buffer contains about 10 mM citrate, about 150 mM NaCl, and a pH of about 4.5. In some embodiments, a suitable mRNA stock solution contains mRNA at a concentration of about 0.10 mg / ml, 1 mg / ml, about 10 mg / ml, about 50 mg / ml, or about 100 mg / ml or higher.

In some embodiments, the citrate buffer is about 100-300 ml / min, 300-
600 ml / min, 600-1200 ml / min, 1200-2400 ml / min, 2400-
Mix at a flow rate in the range of 3600 ml / min, 3600-4800 ml / min, or 4800-6000 ml / min. In some embodiments, the citrate buffer is about 220.
ml / min, about 600 ml / min, about 1200 ml / min, about 2400 ml / min, about 3600 m
Mix at a flow rate of l / min, about 4800 ml / min, or about 6000 ml / min.

In some embodiments, the mRNA stock solution is about 10-30 ml / min, about 30-60 ml.
Mix at flow rates ranging from about 60-120 ml / min, about 120-240 ml / min, about 240-360 ml / min, about 360-480 ml / min, or about 480-600 ml / min. In some embodiments, the mRNA stock solution is about 20 ml / min, about 40 ml / min,
About 60 ml / min, about 80 ml / min, about 100 ml / min, about 200 ml / min, about 300 ml
Mix at a flow rate of about 400 ml / min, about 500 ml / min, or about 600 ml / min.

In some embodiments, the lipid solution contains one or more cationic lipids, one or more helper lipids, one or more cholesterol-based lipids and PEG lipids in ethanol. In some embodiments, the mRNA and lipid solutions are mixed into 20% ethanol.
The result is a suspension of lipid nanoparticles. In some embodiments, the lipid nanoparticles are further purified by tangential flow filtration.

In some embodiments, about 50%, 55%, 60%, 65% of the purified nanoparticles,
70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 9
More than 9% is less than about 100 nm (eg, about 95 nm, about 90 nm, about 85 nm, about 80 n)
m, about 75 nm, about 70 nm, about 65 nm, about 60 nm, about 55 nm, or about 50 nm
Has a size of less than). In some embodiments, substantially all purified nanoparticles are less than 100 nm (eg, about 95 nm, about 90 nm, about 85 nm, about 80 nm, about 7).
It has a size of 5 nm, about 70 nm, about 65 nm, about 60 nm, about 55 nm, or less than about 50 nm).

In some embodiments, about 70%, 75%, 80%, 85% of the purified nanoparticles,
More than 90%, 95%, 96%, 97%, 98%, 99% are about 40-90 nm (eg, about 40-85 nm, about 40-80 nm, about 40-75 nm, about 40-70 nm, about 40- 6
It has a size in the range of 5 nm, or about 40-60 nm). In some embodiments,
Virtually all purified nanoparticles are about 40-90 nm (eg, about 40-85 nm).
It has a size in the range of about 40-80 nm, about 40-75 nm, about 40-70 nm, about 40-65 nm, or about 40-60 nm).

In some embodiments, the purified nanoparticles are about 80%, 85%, 90%, 95%.
Has an encapsulation efficiency greater than 96%, 97%, 98%, or 99%. In some embodiments, the process according to the invention is about 60%, 65%, 70%, 75%, 80%,
It results in the recovery of greater than 85%, 90%, 95%, 96%, 97%, 98%, or 99% mRNA.

In some embodiments, the present invention is a process of encapsulating messenger RNA (mRNA) in lipid nanoparticles, wherein (a) mRNA solution and / or lipid solution.
Separately heating to a predetermined temperature above ambient temperature; (b) mixing heated mRNA and / or heated lipid solution to produce a suspension of lipid nanoparticles; and (c) lipid. Provided is the process, which comprises purifying nanoparticles.

In another aspect, the invention provides a composition of lipid nanoparticles produced by the processes described herein. In some embodiments, the invention is a composition comprising purified lipid nanoparticles, wherein more than about 90% of the purified lipid nanoparticles are less than about 100 nm (eg, about 95 nm, about 90 nm, etc.). About 85 nm, about 80 nm, about 75 nm, about 70 nm, about 65
The composition having a separate particle size (nm, about 60 nm, about 55 nm, or less than about 50 nm), with more than about 70% of the purified lipid nanoparticles encapsulating the mRNA within each individual particle. I will provide a. In some embodiments, about 95% of the purified lipid nanoparticles, 9
6%, 97%, 98%, or more than 99% is less than about 100 nm (eg, about 95 nm, about 90 nm, about 85 nm, about 80 nm, about 75 nm, about 70 nm, about 65 nm, about 60 nm.
, About 55 nm, or less than about 50 nm). In some embodiments, substantially all purified lipid nanoparticles are less than about 100 nm (eg, about 95).
It has individual particle sizes (nm, about 90 nm, about 85 nm, about 80 nm, about 75 nm, about 70 nm, about 65 nm, about 60 nm, about 55 nm, or less than about 50 nm). In some embodiments, about 75%, 80%, 85%, 90%, 95% of the purified lipid nanoparticles.
, 96%, 97%, 98%, or more than 99% encapsulate mRNA within each individual particle. In some embodiments, substantially all purified lipid nanoparticles encapsulate the mRNA within each individual particle. In some embodiments, the compositions according to the invention contain at least about 1 mg, 5 mg, 10 mg, 100 mg, 500 mg, or 1000 mg of encapsulated mRNA.

In some embodiments, each individual lipid nanoparticle comprises one or more cationic lipids, one or more helper lipids, one or more cholesterol-based lipids and PEG lipids. In some embodiments, the one or more cationic lipids are C12-200, MC3, DLinDM.
A, DLinkC2DMA, cKK-E12, ICE (imidazole type), HGT50
00, HGT5001, DODAC, DDAB, DMRIE, DOSPA, DOGS, D
ODAP, DODA and DMDMA, DODAC, DLenDMA, DMRIE, CL
inDMA, CpLinDMA, DMOBA, DOcarbDAP, DLinDAP, D
LincarbDAP, DLinCDAP, KLin-K-DMA, DLin-K-XT
It is selected from the group consisting of C2-DMA, HGT4003, and combinations thereof.

In some embodiments, the one or more non-cationic lipids are DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-).
sn-glycero-3-phosphocholine), DOPE (1,2-diorail-sn-glycero-3-phosphoethanolamine), DOPC (1,2-diorail-sn-glycero-3)
-Phoshotidylcholine) DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE (1,2-dimiristoyl-sn-glycero-3-phosphoethanolamine), DOPG (, 2) -Diole oil-sn-glycero-3-phospho- (1'-rac-glycerol)) is selected.

In some embodiments, the one or more cholesterol-based lipids are cholesterol or PEGylated cholesterol. In some embodiments, one or more PEG modified lipids, a maximum covalently attached to a lipid having a C ₆ -C ₂₀ long alkyl chain (s) 5
Contains poly (ethylene) glycol chains up to kDa.

In some embodiments, the invention is used to encapsulate mRNA containing one or more modified nucleotides. In some embodiments, the invention is used to encapsulate unmodified mRNA.
The present invention provides, for example, the following items.
(Item 1)
The process of encapsulating messenger RNA (mRNA) in lipid nanoparticles, comprising mixing the mRNA solution with the lipid solution, at a predetermined temperature at which the mRNA solution and / or the lipid solution exceeds ambient temperature. There is the process.
(Item 2)
The process of item 1, wherein the predetermined temperature is at or above about 30 ° C, 37 ° C, 40 ° C, 45 ° C, 50 ° C, 55 ° C, 60 ° C, 65 ° C, or 70 ° C.
(Item 3)
The predetermined temperature is in the range of about 25 to 70 ° C., about 30 to 70 ° C., about 35 to 70 ° C., about 40 to 70 ° C., about 45 to 70 ° C., about 50 to 70 ° C., or about 60 to 70 ° C. The process according to item 1 or 2.
(Item 4)
The process according to any one of the preceding items, wherein the predetermined temperature is about 65 ° C.
(Item 5)
The process according to any one of the preceding items, wherein the mRNA solution and the lipid solution are separately heated to the predetermined temperature prior to the mixing.
(Item 6)
The process according to any one of items 1 to 4, wherein the mRNA solution is heated to the predetermined temperature prior to the mixing and the lipid solution is at ambient temperature.
(Item 7)
The process according to any one of the preceding items, wherein the mRNA solution is heated to the predetermined temperature by adding an ambient temperature mRNA stock solution to the heated buffer to bring the mRNA solution to the predetermined temperature.
(Item 8)
7. The process of item 7, wherein the buffer has a pH of about 4.5 or less.
(Item 9)
The process according to any one of the preceding items, wherein the mRNA solution and the lipid solution are mixed by a low pulse flow pump.
(Item 10)
9. The process of item 9, wherein the pump is a gear pump.
(Item 11)
9. The process of item 9, wherein the pump is a centrifugal pump.
(Item 12)
The mRNA solution is in the range of about 150-250 ml / min, 250-500 ml / min, 500-1000 ml / min, 1000-2000 ml / min, 2000-3000 ml / min, 3000-4000 ml / min, or 4000-5000 ml / min. The process according to any one of the preceding items, which is mixed at a flow rate of.
(Item 13)
The preceding item, wherein the mRNA solution is mixed at a flow rate of about 200 ml / min, about 500 ml / min, about 1000 ml / min, about 2000 ml / min, about 3000 ml / min, about 4000 ml / min, or about 5000 ml / min. The process described in any one paragraph.
(Item 14)
The lipid solution is about 25-75 ml / min, about 75-200 ml / min, about 200-350 ml / min, about 350-500 ml / min, about 500-650 ml / min, about 650-850 ml / min, or about 850. The process according to any one of the preceding items, which is mixed at a flow rate in the range of ~ 1000 ml / min.
(Item 15)
The lipid solution is about 50 ml / min, about 100 ml / min, about 150 ml / min, about 200 ml / min, about 250 ml / min, about 300 ml / min, about 350 ml / min, about 400 ml / min, about 450 ml / min, About 500 ml / min, about 550 ml / min, about 600 ml / min, about 650 ml / min, about 700 ml / min, about 750 ml / min, about 800 ml / min, about 850 ml / min, about 900 ml / min, about 950 ml / min, Alternatively, the process according to any one of the preceding items, which is mixed at a flow rate of about 1000 ml / min.
(Item 16)
The process according to any one of the preceding items, wherein the process first produces the mRNA solution by mixing the citrate buffer with the mRNA stock solution.
(Item 17)
The process of item 16, wherein the citrate buffer comprises about 10 mM citrate, about 150 mM NaCl, about 4.5 pH.
(Item 18)
The process of item 16 or 17, wherein the mRNA stock solution comprises the mRNA at a concentration of about 1 mg / ml, about 10 mg / ml, about 50 mg / ml, about 100 mg / ml or higher.
(Item 19)
The citrate buffer is about 100-300 ml / min, 300-600 ml / min, 600-1200 ml / min, 1200-2400 ml / min, 2400-3600 ml / min, 3600-4800 ml / min, or 4800-6000 ml / min. The process of any one of items 16-18, which is mixed at a flow rate in the range of minutes.
(Item 20)
The citrate buffer is mixed at a flow rate of about 220 ml / min, about 600 ml / min, about 1200 ml / min, about 2400 ml / min, about 3600 ml / min, about 4800 ml / min, or about 6000 ml / min. The process according to any one of items 16-19.
(Item 21)
The mRNA stock solution is about 10-30 ml / min, about 30-60 ml / min, about 60-120 ml / min, about 120-240 ml / min, about 240-360 ml / min, about 360-480 ml / min, or about 480. The process of any one of items 16-20, which is mixed at a flow rate in the range of ~ 600 ml / min.
(Item 22)
The mRNA stock solution is about 20 ml / min, about 40 ml / min, about 60 ml / min, about 80 ml / min, about 100 ml / min, about 200 ml / min, about 300 ml / min, about 400 ml / min, about 500 ml / min, Alternatively, the process of any one of items 16-21, which is mixed at a flow rate of about 600 ml / min.
(Item 23)
The process according to any one of the preceding items, wherein the lipid solution comprises one or more cationic lipids, one or more helper lipids, one or more cholesterol-based lipids and PEG lipids in ethanol.
(Item 24)
The process according to any one of the preceding items, wherein the mRNA solution and the lipid solution are mixed into 20% ethanol, resulting in a suspension of lipid nanoparticles.
(Item 25)
24. The process of item 24, wherein the lipid nanoparticles are further purified by tangential flow filtration.
(Item 26)
Approximately 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the purified nanoparticles. 25. The process of item 25, wherein the super has a size of less than 100 nm.
(Item 27)
26. The process of item 26, wherein substantially all of the purified nanoparticles have a size of less than 100 nm.
(Item 28)
Approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, more than 99% of the purified nanoparticles have a size in the range of 50-80 nm. The process according to any one of 25 to 27.
(Item 29)
The process of any one of items 25-28, wherein substantially all of the purified nanoparticles have a size in the range of 50-80 nm.
(Item 30)
Any one of items 25-29, wherein the purified nanoparticles have an encapsulation rate greater than about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%. The process described in.
(Item 31)
The process results in recovery of mRNA greater than about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preceded. The process described in any one of the items.
(Item 32)
A process of encapsulating messenger RNA (mRNA) in lipid nanoparticles, a. Separately heating the mRNA solution and / or lipid solution to a predetermined temperature above ambient temperature;
b. Mixing the heated mRNA solution and / or the heated lipid solution to produce a suspension of lipid nanoparticles; and c. The process comprising purifying the lipid nanoparticles.
(Item 34)
A composition of lipid nanoparticles produced by the process according to any one of the preceding items.
(Item 35)
A composition comprising purified lipid nanoparticles, wherein more than about 90% of the purified lipid nanoparticles have individual particles with a size of less than about 100 nm and about 70 of the purified lipid nanoparticles. % The composition in which the mRNA is encapsulated within each individual particle.
(Item 36)
35. The composition of item 35, wherein about 95%, 96%, 97%, 98%, or more than 99% of the purified lipid nanoparticles have an individual particle size of less than about 100 nm.
(Item 37)
The composition according to any one of items 35 or 36, wherein substantially all of the purified lipid nanoparticles have a separate particle size of less than about 100 nm.
(Item 38)
Approximately 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or more than 99% of the purified lipid nanoparticles encapsulate mRNA within each individual particle. , The composition according to any one of items 35 to 37.
(Item 39)
35. The composition of any one of items 35-38, wherein substantially all of the purified lipid nanoparticles encapsulate mRNA within each individual particle.
(Item 40)
35. The composition of any one of items 35-39, wherein the composition comprises at least 1 mg, 5 mg, 10 mg, 100 mg, 500 mg, or 1000 mg of encapsulated mRNA.
(Item 41)
The composition according to any one of items 35 to 40, wherein each individual lipid nanoparticle comprises one or more cationic lipids, one or more helper lipids, one or more cholesterol-based lipids and PEG lipids. ..
(Item 42)
The one or more cationic lipids are C12-200, MC3, DLinDMA, DLinkC2DMA, cKK-E12, ICE (imidazole type), HGT5000, HGT5001, DODAC, DDAB, DMRIE, DOSPA, DOGS, DODAP, DODMA and DMDMA. , DODAC, DLenDMA, DMRIE, CLINDMA, CpLinDMA, DMOBA, DOcarbDAP, DLinDAP, DLincarbDAP, DLinCDAP, Klin-K-DMA, DLin-K-XTC2-DMA, HGT4003, and a combination thereof. 24. The process or composition according to item 41.
(Item 43)
The one or more non-cationic lipids are DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE (1). , 2- (Giorail-sn-glycero-3-phosphoethanolamine), DOPC (1,2-diorail-sn-glycero-3-phosphotidylcholine) DPPE (1,2-dipalmitoyl-sn-glycero-3) -Phosphoethanolamine), DMPE (1,2-dimiristoyl-sn-glycero-3-phosphoethanolamine), DOPG (, 2-dioreoil-sn-glycero-3-phospho- (1'-rac-glycerol)) The process of item 24 or the composition of item 41, selected from.
(Item 44)
The process according to item 24 or the composition according to item 41, wherein the one or more cholesterol-based lipids are cholesterol or PEGylated cholesterol.
(Item 45)
One or more PEG modified lipids, including C ₆ -C ₂₀ with a maximum covalently attached to a lipid having a length alkyl chain (s) of up to 5kDa length of poly (ethylene) glycol chains, item 24. The process or composition according to item 41.
(Item 46)
The process or composition according to any one of the preceding items, wherein the mRNA comprises one or more modified nucleotides.
(Item 47)
The process or composition according to any one of the preceding items, wherein the mRNA is unmodified.

Other properties, objectives, and advantages of the present invention are evident in the embodiments, drawings, and claims for carrying out the invention below. However, embodiments, drawings, for carrying out the invention,
And while the claims indicate embodiments of the invention, it should be understood that they are given by way of example only, not by limitation. Various changes and modifications will be apparent to those skilled in the art within the scope of the present invention.

The drawings are for illustration purposes only and are not intended to be restrictive.

FIG. 6 shows a schematic diagram of the pharmaceutical process of encapsulating lipid nanoparticles encapsulated mRNA using an exemplary scale using a homogeneous flow pump. An exemplary purification and buffer exchange system for lipid nanoparticles is shown. A schematic diagram of an exemplary scaled-up lipid nanoparticle-encapsulated mRNA formulation process using a peristaltic pump is shown. An alternative exemplary tangential flow filtration system for purification and buffer exchange is shown. An alternative schematic of an exemplary scaled lipid nanoparticle encapsulated mRNA formulation process using a peristaltic pump is shown.

Definitions To make the invention easier to understand, certain terms are first defined below. Additional definitions for the following terms and other terms are specified throughout this specification.

Approximately or Approximately: As used herein, the term "approximately" or "approx."
When applied to one or more values of interest, it refers to a value similar to the stated reference value. In certain embodiments, the terms "approximately" or "about" are used unless otherwise stated or otherwise clear from the context (unless such numbers exceed 100% of possible values). In any direction (greater than or less than) of the stated reference values, 2
5%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 1
Refers to the range of values that fall within 1%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less.

Encapsulation: As used herein, the term "encapsulation" or grammatical equivalent refers to the process of encapsulating individual mRNA molecules within nanoparticles.

Improve, Increase, or Decrease: As used herein, the terms "improve,""increase," or "decrease" or grammatical equivalents are used prior to the initiation of the procedures described herein. Measured values in the same individual, or controls in the absence of the procedures described herein (
Or, the value with respect to the baseline measurement value, such as the measurement value in multiple control objects) is shown.
A "control subject" is a subject who is approximately the same age as the subject to be treated and who has the same form of disease as the subject to be treated.

Impurities: As used herein, the term "impurities" refers to a limited amount of liquid, gas, or solid internal material that differs from the chemical composition of the target material or compound. Impures
Also called a contaminant.

In vitro: As used herein, the term "in vitro" refers to an event that occurs in an artificial environment rather than in a multicellular organism, such as in a test tube or reaction vessel, in a cell culture, and the like.

In vivo: As used herein, the term "in vivo" refers to events that occur within multicellular organisms such as humans and non-human animals. In the context of cell-based systems, the term may be used to refer to events that occur within living cells (eg, as opposed to in vitro systems).

Isolated: As used herein, the term "isolated" refers to (1) the component to which it was associated when first produced (either in a natural and / or experimental setting). Refers to a substance and / or entity that has been isolated from at least some of the above and / or (2) produced, prepared, and / or manufactured by human hands. The isolated substances and / or entities are about 10%, about 20%, about 3 of the other components with which they were initially associated.
0%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 9
It may be separated from 2%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99%. In some embodiments, the isolated agent is about 80.
%, About 85%, About 90%, About 91%, About 92%, About 93%, About 94%, About 95%, About 96
%, About 97%, about 98%, about 99%, or more than about 99% pure. As used herein, a substance is "pure" when it is substantially free of other components. As used herein, the calculation of the purity (%) of an isolated substance and / or entity should not include excipients (eg, buffers, solvents, water, etc.).

Messenger RNA (mRNA): As used herein, "messenger RNA"
The term "(mRNA)" refers to a polynucleotide encoding at least one polypeptide. mRNA includes both modified and unmodified RNA as used herein. The mRNA may contain one or more coding and non-coding regions.

Nucleic Acid: As used herein, the term "nucleic acid", in its broadest sense, refers to any compound and / or substance that is or can be incorporated into a polynucleotide chain. In some embodiments, the nucleic acid is a compound and / or substance that is or can be incorporated into a polynucleotide chain via a phosphodiester bond. In some embodiments, "nucleic acid" refers to individual nucleic acid residues (eg, nucleotides and / or nucleosides). In some embodiments, "nucleic acid" refers to a polynucleotide chain that contains an individual nucleic acid residue. In some embodiments, the "nucleic acid" is
Includes RNA and single-stranded and / or double-stranded DNA and / or cDNA. Furthermore, the terms "nucleic acid", "DNA", "RNA" and / or similar terms include nucleic acid analogs, i.e. analogs having other than the phosphodiester backbone. For example, so-called "peptide nucleic acid" is known in the art and has a peptide bond instead of a phosphodiester bond in the main chain and is considered to be within the scope of the present invention. The term "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are denatured from each other and / or encode the same amino acid sequence. Nucleotide sequences encoding proteins and / or RNA may include introns. Nucleic acids can be purified from natural sources, produced using recombinant expression systems, and optionally purified, chemically synthesized, and the like. Where appropriate, for example, in the case of chemically synthesized molecules, the nucleic acid can include nucleoside analogs such as chemically modified bases or sugars, analogs with backbone modifications and the like. Nucleic acid sequences exist in the 5'to 3'direction unless otherwise specified. In some embodiments, the nucleic acid is a native nucleoside (eg, for example.
Adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosin, deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs (eg, 2-aminoadenosin, 2-thiothymidine, inosin, pyrolo-pyrimidine, 3-methyladenosin, 5-Methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine,
C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C
5-Methylcitidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O (6) -methylguanine, and 2-thiocitidine); chemically modified Modified bases; biologically modified bases (eg, methylated bases); inserted bases; modified sugars (eg, 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexoses ); And / or modified phosphate groups (eg, phosphorothioate and 5'-N-phosphoramidite bonds) or include. In some embodiments, the invention is "not yet" meaning nucleic acids that have not been chemically modified to facilitate or achieve delivery (eg, polynucleotides and residues, including nucleotides and / or nucleosides). It is specifically related to "modified nucleic acid".

Salt: As used herein, the term "salt" refers to an ionic compound that results from or can result from a neutralization reaction between an acid and a base.

Substantially: As used herein, the term "substantially" refers to a qualitative condition exhibiting a characteristic or characteristic of interest in all or almost any degree or degree. Those skilled in the art of biology understand that biological and chemical phenomena, if any, are rarely completed and / or progressed to completion or achieved or avoided absolute consequences. Let's do it. The term "substantially" is therefore used herein to capture the potential lack of integrity inherent in many biological and chemical phenomena.

Yield: As used herein, the term "yield" refers to the percentage of mRNA recovered after encapsulation compared to total mRNA as a starting material. In some embodiments, the term "recovery" is used interchangeably with the term "yield."

The present invention provides an improved process for formulating lipid nanoparticles and encapsulating mRNA. In some embodiments, the present invention is messenger RNA (mRN).
The process of encapsulating A) in lipid nanoparticles, comprising mixing the mRNA solution with the lipid solution, wherein the mRNA solution and / or the lipid solution is at a predetermined temperature above ambient temperature. offer.

Various aspects of the invention are described in detail in the following sections. The use of the term is not intended to limit the invention. Each term can be applied to any aspect of the present invention. In this application, the use of "or" means "and / or" unless otherwise stated.

mRNA
The present invention may be used to encapsulate any mRNA. mRNA is typically considered to be the type of RNA that carries information from DNA to the ribosome. mR
The presence of NA is typically very short, involving processing and translation, followed by decomposition. Typically, in eukaryotes, mRNA processing involves the addition of a "cap" to the N-terminal (5') end and the addition of a "tail" to the C-terminal (3') end. A typical cap is
A 7-methylguanosine cap, which is a guanosine that binds to the first transcribed nucleotide through a 5'-5'-triphosphate bond. The presence of a cap is important in providing resistance to the nuclease found in most eukaryotic cells. The tail is typically a polyadenylation event, which causes the polyadenylation moiety to be mRNA.
It is added to the 3'end of the molecule. The presence of this "tail" is due to the degradation of exonuclease mR
It plays a role in protecting NA. Messenger RNA is translated by the ribosome into a series of amino acids that make up proteins.

mRNA may be synthesized according to any of a variety of known methods. For example, the mRNA according to the present invention may be synthesized via in vitro transcription (IVT). Briefly, IVT is
Typically, a linear or cyclic DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may contain DTT and magnesium ions, and a suitable RNA polymerase (eg, T3, T7, or SP6 RNA polymerase). ), Deoxyribonuclease I, pyrophosphatase, and / or ribonuclease inhibitor.
Strict conditions will vary depending on the specific application.

In some embodiments, the mRNA synthesized in vitro may be purified prior to formulation and encapsulation to remove unwanted impurities including various enzymes and other reagents used during mRNA synthesis. ..

The present invention may be used to formulate and encapsulate mRNAs of various lengths. In some embodiments, the present invention relates to about 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb,
3.5kb, 4kb, 4.5kb, 5kb, 6kb, 7kb, 8kb, 9kb, 10kb
, 11 kb, 12 kb, 13 kb, 14 kb, 15 kb, or 20 kb or more in vitro synthesized mRNA may be used for formulation and encapsulation. In some embodiments, the present invention is about 1-20 kb, about 1-15 kb, about 1-10 kb, about 5-5.
Used to formulate and encapsulate mRNA synthesized in vitro with a length in the range of 20 kb, about 5-15 kb, about 5-12 kb, about 5-10 kb, about 8-20 kb, or about 8-15 kb. good.

The present invention may be used to formulate and encapsulate unmodified mRNA or mRNA containing one or more modifications that typically improve stability. In some embodiments, the modification is selected from modified nucleotides, modified glycophosphate backbone, 5'and / or 3'untranslated regions.

In some embodiments, the modification of the mRNA may include the modification of the nucleotide of the RNA. The modified mRNA according to the present invention can include, for example, backbone modification, sugar modification or base modification. In some embodiments, the mRNA is a purine (adenine (A), guanine (G)) or pyrimidine (thymine (T), cytosine (C), uracil (U)).
And as modified nucleotide analogs or derivatives of purines and pyrimidines, for example, 1-methyl-adenine, 2-methyl-adenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6- Isopentenyl-adenine, 2-
Thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1-methyl-guanine, 2-methyl-guanine, 2,2-
Dimethyl-guanine, 7-methyl-guanine, inosin, 1-methyl-inosin, pseudouracil (5-uracil), dihydro-uracil, 2-thio-uracil, 4-thio-uracil, 5-carboxymethylaminomethyl-2 -Thio-uracil, 5- (carboxyhydroxymethyl) -uracil, 5-fluoro-uracil, 5-bromo-uracil, 5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil, 5-methyl- Uracil, N-uracil-5-oxyacetic acid methyl ester, 5-methylaminomethyl-
Uracil, 5-methoxyaminomethyl-2-thio-uracil, 5'-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 1- Methyl-pseudouracil, cuosin ,.
beta. -D-mannosyl-cuosin, wipe toxosin, and phosphoramic acid,
It may be synthesized from naturally occurring nucleotides and / or nucleotide analogs (modified nucleotides) including, but not limited to, phosphorothioates, peptide nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine and inosine. The preparation of such analogs is described, for example, in US Pat. No. 4,373,071, US Pat. No. 4,401,796, US Pat. No. 4, whose disclosure is incorporated herein by reference in its entirety. , 415,732, US Pat. No. 4,458,066, US Pat. No. 4,50
0,707, US Pat. No. 4,668,777, US Pat. No. 4,973,679, US Pat. No. 5,047,524, US Pat. No. 5,132,418, US Pat. No. 5, 15
It is known to those skilled in the art from 3,319, US Pat. Nos. 5,262,530 and 5,700,642.

Typically, mRNA synthesis involves the addition of a "cap" to the N-terminus (5') and the C-terminus (
3') Includes the addition of a "tail" to the edge. The presence of a cap is important in providing resistance to the nuclease found in most eukaryotic cells. The presence of the "tail" serves to protect the mRNA from exonuclease degradation.

Therefore, in some embodiments, the mRNA comprises a 5'cap structure. A 5'cap is typically added as follows: First, RNA-terminal phosphatase removes one of the terminal phosphate groups from the 5'nucleotide, leaving two terminal phosphates; then guanosine triphosphate. Acid (GTP) is added to terminal phosphate via guanyryl transferase,
It gives rise to a 5'5'5 triphosphate bond: the 7-nitrogen of guanine is then methylated by a methyltransferase. 2'-O-methylation can also occur at the first and / or second base after the 7-methylguanosine triphosphate residue. For example, m7Gp for the cap structure
ppNp-RNA, m7GpppNmp-RNA and m7GppNmpNmp-RNA
(M Refers to a 2'-O methyl residue), but is not limited to these.

In some embodiments, the mRNA comprises a 5'and / or 3'untranslated region. In some embodiments, the 5'untranslated region comprises one or more elements that affect the stability or translation of mRNA, such as iron response elements. In some embodiments, the 5'untranslated region may be about 50-500 nucleotides in length.

In some embodiments, the 3'untranslated region is a polyadenylation signal, the intracellular mR.
Includes one or more binding sites for proteins that affect the stability of NA positions, or one or more binding sites for miRNAs. In some embodiments, the 3'untranslated region is 5
It may be 0 to 500 nucleotides in length or longer.

While the mRNA provided from the in vitro transcription reaction may be desirable in some embodiments, other sources of mRNA are contemplated to be within the scope of the invention, including bacteria, fungi, plants, and /. Or it contains mRNA produced from an animal.

The present invention may be used to formulate and encapsulate mRNAs encoding various proteins. A non-limiting example of mRNA suitable for the present invention is spinal motor neuron 1 (
SMN), alpha-galactosidase (GLA), argininosuccinate synthase (AS)
S1), firefly luciferase, factor IX (FIX), phenylalanine hydroxylase (PAH), and cystic fibrosis membrane conductance receptor (CFTR).
RNA is mentioned. Illustrative mRNA sequences are detailed in the Examples section.

mRNA Solution The mRNA may be provided in a solution that is mixed with the lipid solution so that the mRNA can be encapsulated within the lipid nanoparticles. Suitable mRNA solutions are encapsulated at various concentrations.
It may be any aqueous solution containing RNA. For example, a suitable mRNA solution is mRNA
About 0.01 mg / ml, 0.05 mg / ml, 0.06 mg / ml, 0.07 mg /
ml, 0.08 mg / ml, 0.09 mg / ml, 0.1 mg / ml, 0.15 mg / m
l, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.
6 mg / ml, 0.7 mg / ml, 0.8 mg / ml, 0.9 mg / ml, or 1.0
It may be contained in a concentration of mg / ml or more. In some embodiments, a suitable mRNA solution will produce mRNA at about 0.01-1.0 mg / ml, 0.01-0.9 mg / ml, 0.01.
~ 0.8 mg / ml, 0.01 ~ 0.7 mg / ml, 0.01 ~ 0.6 mg / ml, 0.
01-0.5 mg / ml, 0.01-0.4 mg / ml, 0.01-0.3 mg / ml,
0.01-0.2 mg / ml, 0.01-0.1 mg / ml, 0.05-1.0 mg / m
l, 0.05 to 0.9 mg / ml, 0.05 to 0.8 mg / ml, 0.05 to 0.7 mg
/ Ml, 0.05-0.6 mg / ml, 0.05-0.5 mg / ml, 0.05-0.4
mg / ml, 0.05-0.3 mg / ml, 0.05-0.2 mg / ml, 0.05-0
.. 1 mg / ml, 0.1 to 1.0 mg / ml, 0.2 to 0.9 mg / ml, 0.3 to 0.
It may be contained at a concentration in the range of 8 mg / ml, 0.4 to 0.7 mg / ml, or 0.5 to 0.6 mg / ml. In some embodiments, a suitable mRNA solution will produce mRNA at a maximum of about 5.0 mg / ml, 4.0 mg / ml, 3.0 mg / ml, 2.0 mg / ml, 1.
0 mg / ml ,. 09 mg / ml, 0.08 mg / ml, 0.07 mg / ml, 0.06
It may be contained in concentrations up to mg / ml or 0.05 mg / ml.

Typically, suitable mRNA solutions may also contain buffers and / or salts. Generally, the buffer can include HEPES, ammonium sulfate, sodium hydrogen carbonate, sodium citrate, sodium acetate, potassium phosphate and sodium phosphate. In some embodiments, the preferred concentration of buffer is about 0.1 mM-100 mM, 0.5 mM-90 mM.
, 1.0 mM-80 mM, 2 mM-70 mM, 3 mM-60 mM, 4 mM-50 mM, 5
mM-40 mM, 6 mM-30 mM, 7 mM-20 mM, 8 mM-15 mM, or 9-
It may be in the range of 12 mM. In some embodiments, the preferred concentration of buffer is about 0.
1 mM, 0.5 mM, 1 mM, 2 mM, 4 mM, 6 mM, 8 mM, 10 mM, 15 mM,
It is 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, or 50 mM or more.

Exemplary salts can include sodium chloride, magnesium chloride, and potassium chloride. In some embodiments, the preferred concentration of salt in the mRNA solution is from about 1 mM to 500.
mM, 5 mM to 400 mM, 10 mM to 350 mM, 15 mM to 300 mM, 20 mM to
250 mM, 30 mM to 200 mM, 40 mM to 190 mM, 50 mM to 180 mM, 5
0 mM to 170 mM, 50 mM to 160 mM, 50 mM to 150 mM, or 50 mM to
It may be in the range of 100 mM. The salt concentration in a suitable mRNA solution is about 1 mM, 5 mM,
10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM,
90 mM, or 100 mM or more.

In some embodiments, suitable mRNA solutions are about 3.5-6.5, 3.5-6.0.
, 3.5-5.5. , 3.5-5.0, 3.5-4.5, 4.0-5.5, 4.0-5.5.
0, 4.0-4.9, 4.0-4.8, 4.0-4.7, 4.0-4.6, or 4.0
It may have a pH in the range of ~ 4.5. In some embodiments, a suitable mRNA solution is
Approximately 3.5, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8
4.9, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.1, 6.3, and 6
.. It may have a pH of 5 or less.

Various methods may be used to prepare mRNA solutions suitable for the present invention. In some embodiments, the mRNA may be dissolved directly in the buffers described herein. In some embodiments, the mRNA solution may be produced by mixing the mRNA stock solution with a buffer solution prior to mixing with the lipid solution for encapsulation. In some embodiments, the mRNA solution may be produced by mixing the mRNA stock solution with a buffer solution just prior to mixing with the lipid solution for encapsulation. In some embodiments, a suitable mRNA stock solution is about 0.2 mg.
/ Ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.8 mg / ml,
1.0 mg / ml, 1.2 mg / ml, 1.4 mg / ml, 1.5 mg / ml, or 1
.. 6 mg / ml, 2.0 mg / ml, 2.5 mg / ml, 3.0 mg / ml, 3.5 mg
The mRNA may be contained in water at a concentration of / ml, 4.0 mg / ml, 4.5 mg / ml, or 5.0 mg / ml or higher.

In some embodiments, the mRNA stock solution is mixed with the buffer solution using a pump. Exemplary pumps include, but are not limited to, gear pumps, peristaltic pumps and centrifugal pumps.

Typically, the buffer is mixed in a larger proportion than that of the mRNA stock solution. for example,
The buffer solution is at least 1 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times the ratio of the mRNA stock solution.
It may be mixed in a proportion of times, 8 times, 9 times, 10 times, 15 times, or 20 times larger. In some embodiments, the buffer is about 100-6000 ml / min (eg, about 100-300 m).
l / min, 300-600 ml / min, 600-1200 ml / min, 1200-2400 ml
/ Min, 2400-3600 ml / min, 3600-4800 ml / min, 4800-6000
It may be mixed at a flow rate in the range of ml / min, or 60-420 ml / min). In some embodiments, the buffer is about 60 ml / min, 100 ml / min, 140 ml / min, 180 ml.
/ Min, 220 ml / min, 260 ml / min, 300 ml / min, 340 ml / min, 380 ml
/ Min, 420 ml / min, 480 ml / min, 540 ml / min, 600 ml / min, 1200 m
l / min, 2400 ml / min, 3600 ml / min, 4800 ml / min, or 6000 ml
Mix at a flow rate of / min or higher.

In some embodiments, the mRNA stock solution is about 10-600 ml / min (eg, about 5-5).
50 ml / min, about 10-30 ml / min, about 30-60 ml / min, about 60-120 ml / min, about 120-240 ml / min, about 240-360 ml / min, about 360-480 ml / min,
Alternatively, the mixture is mixed at a flow rate in the range of about 480 to 600 ml / min). In some embodiments, the mRNA stock solution is about 5 ml / min, 10 ml / min, 15 ml / min, 20 ml / min, 25 m.
l / min, 30 ml / min, 35 ml / min, 40 ml / min, 45 ml / min, 50 ml / min, 6
0 ml / min, 80 ml / min, 100 ml / min, 200 ml / min, 300 ml / min, 400
Mix at a flow rate of ml / min, 500 ml / min, or 600 ml / min or higher.

Lipid Solution According to the present invention, the lipid solution contains a mixture of lipids suitable for forming lipid nanoparticles for encapsulating mRNA. In some embodiments, suitable lipid solutions are based on ethanol. For example, a suitable lipid solution is pure ethanol (ie, 100%).
It may contain a mixture of desired lipids dissolved in ethanol). In another embodiment, the suitable lipid solution is based on isopropyl alcohol. In another embodiment, suitable lipid solutions are based on dimethyl sulfoxide. In another embodiment, a suitable lipid solution is
A mixture of suitable solvents including, but not limited to, ethanol, isopropyl alcohol and dimethyl sulfoxide.

Suitable lipid solutions may contain a mixture of desired lipids at various concentrations. For example, suitable lipid solutions are about 0.1 mg / ml, 0.5 mg / ml, 1.0 mg / ml, 2.0 mg /
ml, 3.0 mg / ml, 4.0 mg / ml, 5.0 mg / ml, 6.0 mg / ml, 7
.. 0 mg / ml, 8.0 mg / ml, 9.0 mg / ml, 10 mg / ml, 15 mg / m
l, 20 mg / ml, 30 mg / ml, 40 mg / ml, 50 mg / ml, or 100
It may contain a mixture of desired lipids at a total concentration of mg / ml or greater. In some embodiments, suitable lipid solutions are from about 0.1 to 100 mg / ml, 0.5 to 90 mg / ml, 1.0 to.
80 mg / ml, 1.0-70 mg / ml, 1.0-60 mg / ml, 1.0-50 mg
/ Ml, 1.0-40 mg / ml, 1.0-30 mg / ml, 1.0-20 mg / ml,
1.0 to 15 mg / ml, 1.0 to 10 mg / ml, 1.0 to 9 mg / ml, 1.0 to 8
mg / ml, 1.0-7 mg / ml, 1.0-6 mg / ml, or 1.0-5 mg / m
A mixture of desired lipids may be contained at a total concentration in the range l. In some embodiments, suitable lipid solutions are up to about 100 mg / ml, 90 mg / ml, 80 mg / ml, 70 mg.
/ Ml, 60 mg / ml, 50 mg / ml, 40 mg / ml, 30 mg / ml, 20 mg
It may contain a mixture of desired lipids at a total concentration of up to / ml or 10 mg / ml.

Any desired lipid may be mixed in any proportion suitable for encapsulating the mRNA. In some embodiments, suitable lipid solutions contain a mixture of desired lipids, including cationic lipids, helper lipids (eg, non-cationic lipids and / or cholesterol lipids) and / or PEGylated lipids. In some embodiments, a suitable lipid solution comprises one or more cationic lipids, one or more helper lipids (eg, non-cationic lipids and / or cholesterol lipids) and one or more PEGylated lipids. Contains a mixture of desired lipids.

Cationic Lipids As used herein, the term "cationic lipid" refers to any of the many lipid species that have an effective positive charge at a selected pH, such as physiological pH. Some cationic lipids have been described in the literature and many are commercially available. Cationic lipids that are particularly suitable for use in the compositions and methods of the invention are both incorporated herein by reference in International Patent Publication WO 2010/053572 (and in particular, paragraph [0022].
5] and C12-200) and those described in WO2012 / 170930 are included. In certain embodiments, cationic lipids suitable for the compositions and methods of the invention are provided in US Provisional Patent Application No. 61 / 617,468, filed March 29, 2012.
Ionizable cationic lipids described in No. (incorporated herein by reference), such as (15Z, 18Z) -N, N-dimethyl-6- (9Z, 12Z) -octadeca-9,
12-Diene-1-yl) Tetracosa-15,18-Diene-1-amine (HGT500)
0), (15Z, 18Z) -N, N-dimethyl-6-((9Z, 12Z) -octadeca-
9,12-Diene-1-yl) Tetracosa-4,15,18-Triene-1-amine (H)
GT5001) and (15Z, 18Z) -N, N-dimethyl-6-((9Z, 12Z))
-Octadeca-9,12-diene-1-yl) Tetracosa-5,15,18-triene-
1-Amine (HGT5002) is included.

式中、各Ｒ^２は、独立して、水素またはＣ_１−３アルキルであり；
各ｑは、独立して、２〜６であり；
各Ｒ’は、独立して、水素またはＣ_１−３アルキルであり；
各Ｒ^Ｌは、独立して、Ｃ_８−１２アルキルである、
の化合物またはその医薬的に許容され得る塩を含む。 In some embodiments, cationic lipids suitable for the compositions and methods of the invention include WO201363468 and "Lipid", both of which are incorporated herein by reference.
Forms for Delivery of Messenger R
Includes the cationic lipids described in the US Provisional Patent Application entitled "NA". In some embodiments, the cationic lipids are of formula I-c1-a:

In the formula, each R ² is independently hydrogen or C _1-3 alkyl;
Each q is 2-6 independently;
Each R'is independently hydrogen or C _1-3 alkyl;
Each ^RL is independently _C8-12 alkyl,
Contains the compounds of or pharmaceutically acceptable salts thereof.

In some embodiments, each R ² is independently hydrogen, methyl or ethyl. In some embodiments, each R ² is independently hydrogen or methyl. In some embodiments, each R ² is hydrogen.

In some embodiments, each q is 3-6 independently. In some embodiments, each q is 3-5 independently. In some embodiments, each q is 4.

In some embodiments, each R'is independently hydrogen, methyl or ethyl. In some embodiments, each R'is independently hydrogen or methyl. In some embodiments, each R'is independently hydrogen.

In some embodiments, each ^RL is independently C _8-12 alkyl. In some embodiments, each ^RL is independently n-C _8-12 alkyl. In some embodiments, each ^RL is independently C _9-11 alkyl. In some embodiments,
Each ^RL is independently an n-C _9-11 alkyl. In some embodiments, each ^RL
Is _{independently C 10} alkyl. In some embodiments, each ^RL is independent,
It is n-C ₁₀ alkyl.

In some embodiments, each R ² is independently hydrogen or methyl; each q is independently 3-5; each R'is independently hydrogen or methyl. Each ^RL is independently C _8-12 alkyl.

In some embodiments, each R ² is hydrogen; each q is independently 3-5;
Each R'is hydrogen; each ^RL is independently C _8-12 alkyl.

In some embodiments, each R ² is hydrogen; each q is 4, each R'is hydrogen; each ^RL is independently C _8-12 alkyl.

式中、各Ｒ^Ｌは、独立して、Ｃ_８−１２アルキルである、の化合物またはその医薬的に許
容され得る塩を含む。いくつかの実施形態では、各Ｒ^Ｌは、独立して、ｎ−Ｃ_８−１２ア
ルキルである。いくつかの実施形態では、各Ｒ^Ｌは、独立して、Ｃ_９−１１アルキルであ
る。いくつかの実施形態では、各Ｒ^Ｌは、独立して、ｎ−Ｃ_９−１１アルキルである。い
くつかの実施形態では、各Ｒ^Ｌは、独立して、Ｃ_１０アルキルである。いくつかの実施形
態では、各Ｒ^Ｌは、ｎ−Ｃ_１０アルキルである。 In some embodiments, the cationic lipid is of formula Ig:

In the formula, each ^RL independently _{comprises a compound of C 8-12} alkyl or a pharmaceutically acceptable salt thereof. In some embodiments, each ^RL is independently n-C _8-12 alkyl. In some embodiments, each ^RL is independently C _9-11 alkyl. In some embodiments, each ^RL is independently n-C _9-11 alkyl. In some embodiments, each ^{R L} is, _{independently, C 10} alkyl. In some embodiments, each ^RL is n—C ₁₀ alkyl.

In certain embodiments, suitable cationic lipids are cKK-E12, or (3,6).
-Bis (4- (bis (2-hydroxydodecyl) amino) butyl) piperazine-2,5-
Zeon). The structure of cKK-E12 is shown below:

In some embodiments, one or more cationic lipids suitable for the present invention are N- [1-(
2,3-Diorailoxy) Propyl] -N, N, N-trimethylammonium chloride or "DOTMA" (Feigner et al., (Proc. Nat'l Ac)
ad. Sci. 84,7413 (1987); US Pat. No. 4,897,355). Other suitable cationic lipids include, for example, 5-carboxyspermilglycin dioctadecylamide or "DOGS", 2,3-dioreyloxy-N- [2 (spermine-carboxamide) ethyl] -N, N-. Dimethyl-1-propanaminium or "DOSPA" (
Behr et al., Proc. Nat. 'l Acad. Sci. 86,6982 (1989)
U.S. Pat. No. 5,171,678; U.S. Pat. No. 5,334,761), 1,2-diore oil-3-dimethylammonium-propane or "DODAP", 1,2-diore oil-3-trimethylammonium- Propane or "DOTAP" can be mentioned.

Further exemplary cationic lipids include 1,2-distearyloxy-N, N-dimethyl-3-aminopropane or "DSDMA", 1,2-dioreyloxy-N, N-dimethyl-3-amino. Propane or "DODA", 1,2-dilinoleyloxy-N,
N-Dimethyl-3-aminopropane or "DLinDMA", 1,2-dilinolenyloxy-N, N-dimethyl-3-aminopropane or "DLenDMA", N-diorail-N, N-dimethylammonium chloride or " DODAC, N, N-distearyl-N, N-dimethylammonium bromide or "DDAB"
, N- (1,2-dimyristyloxyprop-3-yl) -N, N-dimethyl-N-hydroxyethylammonium bromide or "DMRIE", 3-dimethylamino-2-
(Cholesta-5-en-3-beta-oxybutane-4-oxy) -1- (cis, cis-
9,12-Octadecadieneoxy) Propane or "CLinDMA", 2- [5'-
(Cholesta-5-en-3-beta-oxy) -3'-oxapentoxy) -3-dimethyl-1- (cis, cis-9', l-2'-octadecadieneoxy) propane or "C
pLinDMA ", N, N-dimethyl-3,4-diorailoxybenzylamine or" DMOBA ", 1,2-N, N'-diorail carbamil-3-dimethylaminopropane or" DOcarbDAP ", 2,3 -Dilinole oil oxy-Ν, Ν-dimethylpropylamine or "DLinDAP", 1,2-N, N'-dilinoleyl carbamil-
3-Dimethylaminopropane or "DLincarbDAP", 1,2-dilinole oil carbamil-3-dimethylaminopropane or "DLinCDAP", 2,2-dilinoleyl-4-dimethylaminomethyl- [1,3] -dioxolane or "DLin ---
DMA ", 2,2-dilinoleyl-4-dimethylaminoethyl- [1,3] -dioxolane or" DLin-K-XTC2-DMA ", and 2- (2,2-di ((9Z, 12Z))
) -Octadeca-9,12-diene-1-yl) -1,3-dioxolane-4-yl)-
N, N-dimethylethaneamine (DLin-KC2-DMA)) (WO2010 / 042)
877; Simple et al., Nature Biotechnology. 28: 172-176 (20)
10)) or mixtures thereof (Heies, J. et al., J. et al.
Controlled Release 107: 276-287 (2005); Mo
Rrissey, DV. Et al., Nat. Biotechnol. 23 (8): 1003-1
007 (2005); PCT Publication WO 2005/121348A1). In some embodiments, one or more of the cationic lipids comprises at least one of an imidazole, dialkylamino, or guanidium moiety.

In some embodiments, the one or more cationic lipids are XTC (2,2-dilinoleyl-4-dimethylaminoethyl- [1,3] -dioxolane), MC3 (((6Z, 9Z)).
, 28Z, 31Z) -Heptatria Conta-6,9,28,31-Tetraene-19-yl4- (dimethylamino) butanoate), ALNY-100 ((3aR, 5s, 6aS)
) -N, N-dimethyl-2,2-di ((9Z, 12Z) -octadeca-9,12-dienyl) tetrahydro-3aH-cyclopenta [d] [1,3] dioxol-5-amine)
), NC98-5 (4,7,13-tris (3-oxo-3- (undecylamino) propyl) -N1, N16-diundecyl-4,7,10,13-tetraazahexadecane-
1,16-Diamide), DODAP (1,2-diorail-3-dimethylammonium propane), HGT4003 (WO2012 / 170888, the teachings of which are incorporated herein by reference in their entirety), ICE (WO2011 / 068810). , The teachings of which are incorporated herein by reference in their entirety), HGT5000 (US Provisional Patent Application No. 61 / 617,468, the teachings of which are incorporated herein by reference in their entirety) or HGT5001. (Sys or Trans) (Temporary Patent Application No. 61 / 617,468
No.), aminoalcohol lipidoids such as those disclosed in WO2010 / 053572, DOTAP (1,2-diorail-3-trimethylammonium propane), DO
TMA (1,2-di-O-octadecenyl-3-trimethylammonium propane), D
LinDMA (Heies, J .; Palmer, L .; Bremner, K .; Mac
Lachlan, I. et al. “Cationical Lipid Saturation inf
lunaces intracellular delivery of encaps
qualified nucleic acids "J. Control. Rel. 2005, 10
7,276-287), DLin-KC2-DMA (Semple, SC et al., "Ra"
torsional Design of Chemical Lipids for siR
NA Delivery "Nature Biotechnology. 2010, 28, 172-1
76), C12-200 (Love, KT et al., "Lipid-like mater"
ials for low-dose in vivo gene silencing
It may be selected from "PNAS 2010, 107, 1864-1869".

In some embodiments, the cationic lipid is at least about 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50 of the total lipid in a suitable lipid solution by weight or molar. %
, 55%, 60%, 65%, or 70%. In some embodiments, the cationic lipid (s) are about 30-70% (eg, about 30-65%, about 30-60%, about 30-55%) of the total lipid mixture by weight or mole. , About 30-50%, about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%).

Non-cationic / helper lipids As used herein, the term "non-cationic lipid" refers to any neutral, zwitterionic or anionic lipid. As used herein, the phrase "anionic lipid" refers to any of the many lipid species that have a net negative charge at a selected pH, such as physiological pH. Non-cationic lipids include distearoylphosphatidylcholine (DSPC), dioleoil phosphatidylcholine (DOPC), and dipalmitoylphosphatidylcholine (DP).
PC), Dioleoil Phosphatidylglycerol (DOPG), Dipalmitoylphosphatidylglycerol (DPPG), Dioleoil Phosphatidylethanolamine (D)
OPE), palmitoyl ole oil phosphatidylcholine (POPC), palmitoyl ole oil-phosphatidylethanolamine (POPE), diore oil-phosphatidylethanolamine 4- (N-maleimidemethyl) -cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyle Phosphatidylethanolamine (DPPE)
), Dimiristylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-ole Contains, but is not limited to, oil-phosphatidylethanolamine (SOPE), or mixtures thereof.

In some embodiments, the non-cationic lipid is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35% of the total lipid in a suitable lipid solution by weight or molar. 40
%, 45%, 50%, 55%, 60%, 65% or 70% may be composed. In some embodiments, the non-cationic lipid (s) are about 30-50% (eg, about 30-45%, about 30-40%) of the total lipid in a suitable lipid solution by weight or molar. , About 35-50%,
It constitutes about 35-45%, or about 35-40%).

Cholesterol-based lipids In some embodiments, a suitable lipid solution comprises one or more cholesterol-based lipids. For example, suitable cholesterol-based cationic lipids include, for example, DC-Choi (N, N).
-Dimethyl-N-ethylcarboxamide cholesterol), 1,4-bis (3-N-oleylamino-propyl) piperazine (Gao et al., Biochem. Biophys. Re
s. Comm. 179,280 (1991); Wolf et al., BioTechniques
23,139 (1997); US Pat. No. 5,744,335), or ICE. In some embodiments, the cholesterol-based lipids (s) are at least about 5%, 10%, 20%, 30%, 40%, 50% of the total lipids in a suitable lipid solution by weight or molarity.
, 60%, or 70%. In some embodiments, cholesterol-based lipids (
Multiple) by weight or mol is about 30-50% of the total lipid in a suitable lipid solution (eg, for example).
About 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-4
0%).

PEGylated Lipids In some embodiments, a suitable lipid solution comprises one or more PEGylated lipids.
Polyethylene glycol (PEG) modified phospholipids and derivatized lipids, such as
Derivatized ceramide (C8PEG-2000 ceramide) containing N-octanoyl-sphingosine-1- [succinyl (methoxypolyethylene glycol) -2000] (C8PEG-2000 ceramide)
The use of PEG-CER) is also contemplated by the present invention. Lipids PEG-modified are contemplated, including polyethylene glycol chain length up 5kDa a maximum of covalent attachment to a lipid with C ₆ -C ₂₀ long alkyl chain (s) is not limited to this. In some embodiments, the PEG-modified or PEGylated lipid is PEGylated cholesterol or PEG-2.
It is K. In some embodiments, a particularly useful exchangeable lipid is the shorter acyl chain (
For example, PEG-ceramide having _{C 14} or C _18).

PEG-modified phospholipids and derivatized lipids, by weight or molar, are at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, or of total lipids in a suitable lipid solution. It may constitute 70%. In some embodiments, the PEGylated lipid lipid (s) is about 30-50% (eg, about 30-45%) of the total lipid in a suitable lipid solution by weight or molar.
, About 30-40%, about 35-50%, about 35-45%, or about 35-40%).

Illustrative combinations of cationic lipids, non-cationic lipids, cholesterol-based lipids, and PEG-modified lipids are described in the Examples section. For example, a suitable lipid solution is cKK
-E12, DOPE, chol, and DMG-PEG2K; C12-200, DOPE,
Cholesterol and DMG-PEG2K; HGT5000, DOPE, chol, and DMG-PEG2K; HGT5001, DOPE, chol, and DMG-PEG2K;
cKK-E12, DPPC, chol, and DMG-PEG2K; C12-200, DP
PC, cholesterol, and DMG-PEG2K; HGT5000, DPPC, chol
, And DMG-PEG2K; or HGT5001, DPPC, chol, and DMG-
It may contain PEG2K. The selection of cationic lipids, non-cationic lipids, and / or PEG-modified lipids, including lipid mixtures, and the relative molar ratio of such lipids to each other are characteristics of the selected lipid (s), as well as Based on the characteristics and properties of the encapsulated mRNA. Further considerations include, for example, saturation of the alkyl chain, as well as the size, charge, pH, pKa, membrane fusion, and toxicity of the selected lipid (s). Therefore, the molar ratio may be adjusted as appropriate.

Mixing Process The present invention is based on the discovery of the unexpected effect of temperature on mRNA encapsulation efficiency and recovery. Therefore, in some embodiments, the present invention presents the messenger RNA (mRNA) in lipid nanoparticles by mixing the mRNA and lipid solutions described herein.
The process of encapsulating the mRNA solution and / or the lipid solution is provided, wherein the mRNA solution and / or the lipid solution is heated to a predetermined temperature exceeding the ambient temperature. As used herein, the term "ambient temperature" refers to room temperature, or the temperature surrounding an object of interest (eg, mRNA or lipid solution) without heating or cooling. In some embodiments, the ambient temperature refers to a temperature in the range of about 20-25 ° C.

Therefore, a given temperature above the ambient temperature typically exceeds about 25 ° C. In some embodiments, the preferred temperatures for the present invention are about 30 ° C, 37 ° C, 40 ° C, 45 ° C, 50.
° C., 55 ° C., 60 ° C., 65 ° C., or 70 ° C. or higher. In some embodiments, the preferred temperatures for the present invention are about 25-70 ° C, about 30-70 ° C, about 35-70 ° C, about 40-.
It is in the range of 70 ° C., about 45-70 ° C., about 50-70 ° C., or about 60-70 ° C. In certain embodiments, the preferred temperature for the present invention is about 65 ° C.

The mRNA solution, the lipid solution, or both may be heated to a predetermined temperature above ambient temperature prior to mixing. In some embodiments, the mRNA and lipid solutions are separately heated to a predetermined temperature prior to mixing. In some embodiments, the mRNA and lipid solutions are mixed at ambient temperature, then heated to a predetermined temperature after mixing. In some embodiments, the lipid solution is heated to a predetermined temperature and mixed with the mRNA solution at ambient temperature. In some embodiments, the mRNA solution is heated to a predetermined temperature and mixed with the lipid solution at ambient temperature.

In some embodiments, the mRNA solution is heated to a predetermined temperature by adding an ambient temperature mRNA stock solution to a heated buffer to achieve the desired predetermined temperature.

The mRNA solution and the lipid solution may be mixed using a pump. The encapsulation procedure is
Since it can occur on a wide range of scales, different types of pumps may be used to adapt to the desired scale. However, it is generally desirable to use a low pulse flow pump. As used herein, a low pulse flow pump refers to any pump that can establish a continuous flow at a stable flow rate. Suitable pump types are, but are not limited to,
Includes gear pumps and centrifugal pumps. Exemplary gear pumps include, but are not limited to, Core-Parmer or Diener gear pumps. Exemplary centrifugal pumps include, but are not limited to, Grainger or Core-Parm.
Includes those manufactured by er.

The mRNA solution and the lipid solution may be mixed at various flow rates. Typically, the mRNA solution may be mixed in a proportion greater than that of the lipid solution. For example, mRNA solution is at least 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x that of lipid solution.
It may be mixed in a proportion of times, 15 times, or 20 times larger.

The flow rate suitable for mixing may be determined based on the scale. In some embodiments, mR
NA solution is about 40-400 ml / min, 60-500 ml / min, 70-600 ml / min,
80-700 ml / min, 90-800 ml / min, 100-900 ml / min, 110-10
00 ml / min, 120-1100 ml / min, 130-1200 ml / min, 140-130
0 ml / min, 150-1400 ml / min, 160-1500 ml / min, 170-1600
ml / min, 180-1700 ml / min, 150-250 ml / min, 250-500 ml / min
Minutes, 500-1000 ml / min, 1000-2000 ml / min, 2000-3000 ml
Mix at a flow rate in the range of / min, 3000-4000 ml / min, or 4000-5000 ml / min. In some embodiments, the mRNA solution is about 200 ml / min, about 500 m.
l / min, about 1000 ml / min, about 2000 ml / min, about 3000 ml / min, about 4000 m
Mix at a flow rate of l / min or about 5000 ml / min.

In some embodiments, the lipid solution is about 25-75 ml / min, 20-50 ml / min,
25-75 ml / min, 30-90 ml / min, 40-100 ml / min, 50-110 ml / min
Minutes, 75-200 ml / min, 200-350 ml / min, 350-500 ml / min, 500
Mix at a flow rate in the range of ~ 650 ml / min, 650-850 ml / min, or 850-1000 ml / min. In some embodiments, the lipid solution is about 50 ml / min, about 100 m.
l / min, about 150 ml / min, about 200 ml / min, about 250 ml / min, about 300 ml / min,
About 350 ml / min, about 400 ml / min, about 450 ml / min, about 500 ml / min, about 550
ml / min, about 600 ml / min, about 650 ml / min, about 700 ml / min, about 750 ml / min, about 800 ml / min, about 850 ml / min, about 900 ml / min, about 950 ml / min, or about 1000 ml / min. Mix at flow rate.

Typically, the mRNA solution and the lipid solution are mixed in the solution so that the lipid can form nanoparticles that encapsulate the mRNA. Such a solution is also referred to as a formulation or an encapsulated solution. Suitable formulations or encapsulated solutions may be based on a solvent such as ethanol. For example, a suitable formulation or encapsulating solution may be based on about 10% ethanol, about 15% ethanol, about 20% ethanol, about 25% ethanol, about 30% ethanol, about 35% ethanol, or about 40% ethanol. ..

Suitable formulations or encapsulated solutions may be based on a solvent such as isopropyl alcohol. For example, a suitable formulation or encapsulating solution is about 10% isopropyl alcohol, about 15% isopropyl alcohol, about 20% isopropyl alcohol, about 25% isopropyl alcohol, about 30% isopropyl alcohol, about 35% isopropyl alcohol, or about 40. % Isopropyl alcohol may be the base.

Suitable formulations or encapsulated solutions may be based on solvents such as dimethyl sulfoxide. For example, a suitable formulation or encapsulating solution is about 10% dimethyl sulfoxide, about 15% dimethyl sulfoxide, about 20% dimethyl sulfoxide, about 25% dimethyl sulfoxide, about 30% dimethyl sulfoxide, about 35% dimethyl sulfoxide, or about 40.
% Dimethyl sulfoxide may be the base.

Suitable formulations or encapsulated solutions may also contain buffers or salts. An exemplary buffer may include HEPES, ammonium sulfate, sodium hydrogen carbonate, sodium citrate, sodium acetate, potassium phosphate and sodium phosphate. Exemplary salts may include sodium chloride, magnesium chloride, and potassium chloride.

Purification Typically, after formulation and encapsulation, the lipid nanoparticles are purified and / or concentrated. Various purification techniques may be used. In some embodiments, the lipid nanoparticles are purified using tangential flow filtration. Tangent flow filtration (TFF), also referred to as cross-flow filtration, is a type of filtration in which the material to be filtered passes tangentially rather than through the filter. In TFF, unwanted permeated water passes through the filter, while the desired object to be retained passes along the filter and is collected downstream. It is important to note that in TFF, the desired material is typically contained in the payload, as opposed to what is normally encountered in conventional dead-end filtration.

Depending on the material being filtered, TFF is used for either microfiltration or ultrafiltration. Microfiltration is typically defined as an example where the filter has a pore size between 0.05 μm and 1.0 μm, including both ends, while ultrafiltration is typically defined.
It necessarily includes a filter with a pore size of less than 0.05 μm. Pore size is the nominal fractional molecular weight (NMWL), also known as the molecular weight cutoff (MWCO) for a particular filter.
Also determined, typically microfiltration membranes exceed 1,000 kilodaltons (kDa) NM
It has a WL and the ultrafiltration filter has an NMWL between 1 kDa and 1,000 kDa.

The main advantage of tangential flow filtration is that impermeable particles (sometimes also referred to as "filtration cakes") that can aggregate and block the filter during conventional "dead end" filtration, instead of the surface of the filter. Is to be carried along. This advantage generally eliminates the need to remove and clean the filter, thus significantly reducing work interruption time, allowing tangential flow filtration to be widely used in industrial processes requiring continuous operation.

Tangential flow filtration can be used for several purposes, including concentration and diafiltration, among others. Concentration is the process by which the solvent is removed from the solution while retaining the solute molecules. To concentrate the sample efficiently
Membranes with NMWL or MWCO substantially lower than the molecular weight of the retained solute molecule are used. Generally, one of ordinary skill in the art may select a filter having an NMWL or MWCO that is 3 to 6 times less than the molecular weight of the target molecule (s).

Diafiltration allows small unwanted particles to pass through the filter, while large desired nanoparticles do not change the concentration of the nanoparticles in solution.
A fractionation process that is maintained in the object to be held. Diafiltration is often used to remove salts or reaction buffers from solution. Diafiltration is
It may be continuous or discontinuous. In continuous diafiltration, the diafiltration solution is added to the sample at the same rate at which the filtrate is formed. In discontinuous diafiltration, the solution is first diluted and then concentrated back to the starting concentration. Discontinuous diafiltration may be repeated until the desired concentration of nanoparticles is reached.

Purified and / or concentrated lipid nanoparticles may be formulated in a desired buffer, such as PBS.

Providing Nanoparticles for Encapsulating mRNA The process according to the invention is more homogeneous and smaller particle size (eg, less than 100 nm) and significantly improved encapsulation efficiency and / or It results in mRNA recovery.

Therefore, the present invention provides a composition comprising the purified nanoparticles described herein. In some embodiments, the majority of the purified nanoparticles in the composition, ie, about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85 of the purified nanoparticles. %, 90%
, 95%, 96%, 97%, 98%, or more than 99% is less than about 100 nm (eg, about 95 nm, about 90 nm, about 85 nm, about 80 nm, about 75 nm, about 70 nm, about 65 nm.
, About 60 nm, about 55 nm, or less than about 50 nm). In some embodiments, substantially all purified nanoparticles are less than 100 nm (eg, about 95 nm).
, About 90 nm, about 85 nm, about 80 nm, about 75 nm, about 70 nm, about 65 nm, about 60
It has a size of nm, about 55 nm, or less than about 50 nm).

In addition, more homogeneous nanoparticles with a narrow nanoparticle size range are achieved by the process of the present invention. For example, about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, more than 99% of the purified nanoparticles in the compositions provided by the present invention. , About 40-90 nm (eg, about 40-85 nm, about 40-80 nm, about 40-75 n
It has a size in the range (m, about 40-70 nm, about 40-65 nm, or about 40-60 nm). In some embodiments, substantially all purified nanoparticles are about 40-90.
nm (eg, about 40-85 nm, about 40-80 nm, about 40-75 nm, about 40-70
It has a size in the range of nm, about 40-65 nm, or about 40-60 nm).

In some embodiments, the dispersibility of nanoparticles in the compositions provided by the present invention, or a measure of homogeneity in molecular size (PDI), is less than about 0.16 (eg, about 0.1).
5, 0.14, 0.13, 0.12, 0.11, 0.10, 0.09, or less than 0.08).

In some embodiments, about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or of the purified lipid nanoparticles in the compositions provided by the present invention. 99%
Super encapsulates mRNA within each individual particle. In some embodiments, substantially all purified lipid nanoparticles in the composition encapsulate the mRNA in each individual particle.

In some embodiments, the compositions according to the invention are at least about 1 mg, 5 mg, 10
Contains mg, 100 mg, 500 mg, or 1000 mg of encapsulated mRNA. In some embodiments, the process according to the invention is about 60%, 65%, 70%,
It results in the recovery of greater than 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% mRNA.

While certain compounds, compositions and methods of the present invention have been described with specificity according to certain particular embodiments, the following examples serve merely to illustrate the compounds of the present invention. Not intended to be restricted.

Example 1. Effect of Temperature on Nanoparticle Encapsulation Process This example demonstrates that an increase in temperature during the nanoparticle encapsulation process results in an increase in yield and / or encapsulation efficiency.

Lipid Materials Unless otherwise specified, the formulations described in the Examples below are one or more cationic lipids, helper lipids (eg, non-cationic lipids and, etc.) designed to encapsulate various nucleic acid materials. / Or contains a multi-component lipid mixture in various proportions that employ cholesterol lipids) and PEGylated lipids. Cationic lipids for this process are DOTAP (1,2-
Dioleyl-3-trimethylammonium propane), DODAC (1,2-diorail-3-dimethylammonium propane), DOTMA (1,2-di-O-octadecenyl-3-trimethylammonium propane), DLinDMA (Heies, J .; Pal
mer, L. Bremner, K. et al. MacLachlan, I. et al. "Cationic
lipid saturation influences intracellul
ar delivery of augmented nucleoic acid
s "J. Control. Rel. 2005, 107, 276-287), DLin-KC2
-DMA (Semple, SC et al., "Rational Design of Ca"
torsion Lipids for siRNA Delivery "Nature"
Biotechnology. 2010, 28, 172-176), C12-200 (Love, K. et al.
T. Et al., "Lipid-like materials for low-dose i"
n vivo gene silencing "PNAS 2010, 107, 1864
-1869), cKK-E12 (3,6-bis (4- (bis (2-hydroxydodecyl))
Amino) Butyl) Piperazine-2,5-dione), HGT5000, HGT5001, H
GT4003, ICE, dialkylamino type, imidazole type, guanidium type and the like can be included, but the present invention is not limited thereto. Helper lipids include DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) and DPPC (1,2-dipalmitoyl-s).
n-glycero-3-phosphocholine), DOPE (1,2-diorail-sn-glycero-)
3-phosphoethanolamine), DOPC (1,2-diorail-sn-glycero-3-3)
Phosphotidylcholine) DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE (1,2-dimiristoyl-sn-glycero-3-phosphoethanolamine), DOPG (, 2- Diole oil-sn-glycero-3-phospho-
(1'-rac-glycerol)), cholesterol and the like can be included, but are not limited thereto. The PEGylated lipid can include, but is not limited to, a C6-C20 length alkyl chain (s) and a poly (ethylene) glycol chain up to 5 kDa co-adhering to the lipid.

Messenger RNA Material Codon-Optimized Human Spinal Motor Neuron 1 (SMN) Messenger RNA, Argininosuccinate Synthesizer (ASS1) Messenger RNA, Modified Cystic Fibrosis Membrane Conductance Regulator (SNIM® CFTR, 25% Pseudouridine, 25) % 5-Methyl-citidine) messenger RNA, firefly luciferase (FFL) messenger RNA, factor IX (FIX) messenger RNA, phenylalanine hydroxylase (PAH) messenger R
NA and alpha-galactosidase (GLA) messenger RNA were synthesized by in vitro transcription from a plasmid DNA template encoding the gene, followed by a 5'cap structure (Cap 1) (Fechter, P .; Brownlee, GG ". Recog
nition of mRNA cap strands by viral a
nd cellular protein "J. Gen. Virology 2005
, 86, 1239-1249) and a 3'poly A tail with a length of approximately 250 nucleotides as determined by gel electrophoresis was added. 5'and 3 present in each mRNA product
'Untranslated regions are represented as X and Y, respectively, and are defined as described (below).
Codon-optimized human spinal motor neuron 1 (SMN) mRNA:
XAUGGCCAUGAGGACAGCGGAGGCAGCGGCGGGGAGUGCCC
GAGCAGGAGGAGAGCGUGCUGUUCAGGAGAGGCACCGGCC
AGAGCGAUGACAGGCGAUAUCUGGGAGACGAUCCGCUGUGAU
CAAGGCCUACGACAAGGCCCGUGGCCAGCUUCAAGCACCGCC
CUGAAAAACGGGCGACAUCCUGCGAGAGACCAGCGCAAGCCCA
AGACAACCCCCAAGAGAAAAGCCCGCCAAGAAGAAUAAGAG
CCAGAAAAAGAACACCGCCGCCAGCCUGCAGCAGUGGAAG
GUGGGGCGACAAGUGCAGGCCAUCCUGGGAGCGAGGACGGCU
GCAUCUCCCCGCCACCAUCGCCAGCAUCGACUUCAAGAG
AGAGACCUGCGUGGUCGUGUACACCGGCUACGGCAACAGA
GAGGAGCAGACGUGAGCGACCUGCUGAGCCCCUUGUG
AGGUGGCCAAUAACAUCGAACAGAACGCCAGGGAGAACGA
GAAUGAAAGCCAGGUGAGCACCGACGAGAGCGAGAACAGC
AGAUCUCCUGGCAACAAGAGCGACAACAUCAAGCCUAAGU
CUGCCCCUGGAACAGCUUCCUGCCCCCUCCUCCACCAU
GCCCGGACCCAGACUGGGACCCGGAAAACCUGGCCUGAAG
UUCAACGGACCACCUCCCCCUCACCUCCUCCUCCCACCUCUC
AUCUCCUGAGCUGCUGCGCUGCCCUUCCUCCAGCGACC
CCCUAUCAUCCCACCACCCCCCUCCUCUCUGCCCCGACAGC
CUGGACGACCGCCGAUGCCCCUGGGGCAGGCAUGCUGAUCAGCU
GGUACAUGAGCGGCUACCACACACAGGAUACUACAUAGUCGCUU
CAGACAGAACCAGAAGGAGGGGCAGAUGCUCCCACUCCCUG
AACUGAY
Human alpha-galactosidase (GLA) mRNA:
XAUGCAGCUGAGGGAACCCAGAACUACAUCUGGGGCUGCCGCG
CUUGCGCUUCCGCUUCCUGGGCCCUCGUUUCCUGGGACAUCC
CUGGGCGCUAGAGCACUGGACAAUGGAGAUGGCAAGGACGCC
UACCAUGGCGCUGGCUGCACUGGGGAGCGGCUUCAUGUGCAAC
CUUGACUGCCAGGAAGAGCCAGAUUCCUGCAUCAGUGAGA
AGCUCUUCAUGGAGAGAGAGCAGAGGCUCAUGGUCUCAGAAGG
CUGGAAGGAUGCAGGGUUAUGAGUACCUCUGCAUGAUGAC
UGUUGGAUGGCUCCCCAAAGAGAUUCAGAGCAGACUUC
AGGCAGACCCUCAGGCGCUUUCCUCAUGGAUUCCGCCAGCU
AGCUAAUUAUGUUCACAGCAAAAGGAGUGAAGCUAGGGAUU
UAUGCAGAUGUGUGGAAAAUAAAAACCUGCGCAGGCUUCCCUG
GGAGUUUGGAUACUACGUACAUUGAUGCCCAGACCUUGC
UGACUGGGGAGUAGAUCUGCUAAAAAUUGAUGAUGGUUGUUC
UGUGACAGUUGGAAAAUUUGCAGAUGGUUAAGCACA
UGUCCUUGGCCUGAAUAGGACUGGCAGAAGCAUGUGUA
CUCCUGUGAGUGGCCUCCUUUAUAUGUGCGCCCUUUCAAAG
CCCAAUUAUAGAAAAUCCGACAGUACUGCAAUCACUGGC
GAAAUUUGCUGACAUGAUGAUGAUUCCUGGGAAAAGUAUAAA
GAGUAUCUUGGACUGGACAUCUUUAACCAGGAGAGAAUU
GUUGAUGUUGCUGGACCAGGGGGUUGGAAUGACCCAGAUA
UGUUAGUGAUGGCAACUUGGCCUCAGCUGGAAUCAGCA
AGUAACUCAGAUGGCCCUCUGGGGCUAUCAUGCGCUGCUCCU
UUAUUCAUGUCUAAAUGACCUCCGACAUCAGCCCUCAAG
CCAAAGCUCUCCUCUCAGGAUAAGGAAGUAAUGCCAUCAA
UCAGGACCCCUUGGGCAAGCAAGGGGUACCAGCUUAGACAG
GGAGACAACUUUGAGUGUGGGAACGACCUCUCUCAGGCU
UAGCCUGGGCUGUAGCUAUGAUAAACCGGCAGGGAGAUGG
UGGACCUCCGCUCUUAUAUCCAUCCAGUUGCUUCCUCGGU
AAAGGAGUGGCCUGUAAAUCCUGCCUGCUUCACACAGC
UCCUCCCUGUGAAAAGGAAGCUAGGGGUUCUAUGAUGGAC
UUCAAGGUUAGAAGUCACAUAAAUCCCACAGGCACUGUU
UUGCUUCAGCUAGAAAAAUACAAUGCAGAUGUCAUUAAAAG
ACUUACUUUAY
Codon-optimized human argininosuccinate synthase (ASS1) mRNA:
XAUGAGCAGCAAGGGCAGCGUGGUGCUGGCCUACAGCGGC
GGCCUGGACACCAGCUGCAUCCUGGUGGCUGAAGGAGC
AGGGCUACGAGUGAUCCGCCUACCUGCCAAACAGGCCA
GAAGGAGGACUUCGAGGGAGGCCCGCAAGAAGGCCCUGAAG
CUGGGCGCCAAGAAGGUGUUCAUCCGAGGACGUGAGCCGCG
AGUUCGUGGAGGAGUGUCAUCUGGGCCGCCAUCCAGAGCAG
CGCCCUGUACGAGGACCGCUACCUGCUGGGCACCAGCCUG
GCCCGCCCCUGCAUCCGCCCGCAAGCAGGUGGAGAUCGCCC
AGCGCGAGGGGCGCCAAGUACGUGAGCCACGGCGCCACCGG
CAAGGGCAACGACCAGGUGCCGCUUCGAGCUGAGCUGCUAC
AGCCUGGCCCCCCAGAUCAAGGUGAUCGCCCCCUGCGCA
UGCCCGAGUUCUAACCGCUUCAUCAAGGGCCGCAACCGU
GAUGGAGUACGCCAAGCAGCACGGCAUCCCCAUCCCCGUG
ACCCCCAAGAACCCCUGGAGCAUGGACGAGAACCUGAC
ACAUCAGCUACGAGGCCGGCAUCCUGGGAGAACCCAAGAA
CCAGGCCCCCCCCGGCCUGUACACCAAGACCCCAGGACCCC
GCCAAGGCCCCAACACCCCCCGACAUCCUGGGAGAUCGAGU
UCAAGAAGGGGCGUGCCCGUGAAGGUGACCAACGUGAAGGA
CGGCACCACCACCACCAGACCAGCCUGGAGCUGUUCAUGUAC
CUGAACGAGGUGCGCCGGCAAGCACGGGCGGUGGGCCGCAUCG
ACAUCGUGGAGAACCGCUUCAUCGGCAUGAAGAGCCGGCG
CAUCUCGAGACCCCCGCCGGCACCAUCCUGUACCACCC
CACCUGGACAUCGAGGCCUUCACCAUGGACCGGCGAGGUGC
GCAAGAUCAAGCAGGCGCCUGGGCCUGAGUUCCGCCGAGCU
GGUGUACACCGGCUUCUGGCACAGCCCCGAGUGCGAGUUC
GUGCGCCACUGCAUCCGCCAAGAGCCAGGAGCGGCGGGGAGG
GCAAGGUGCAGGUGAGCGUGGCUGAAGGGCCAGGUGUACAU
CCUGGGCCGCGGAGAGCCCCCUGAGCCUGUACAAACGAGGAG
CUGGUGAGCAUGAACGUGCAGGGGCGACUACGAGCACCCG
ACGCCACCGGCUUCAUCAACAUCAACAGCCUGCGCCUGAA
GGAGUACCACCGCCUGGACAGCAAGGUGACCGCCAAGUGA
Y
Codon-optimized firefly luciferase mRNA:
XAUGGAAGAUGCCAAAAACAUUAGAAGGGCCCAGCGCCA
UUCUCCCACUCGAAGACGGGACCGGCCGGGCAGCAGCUGC
ACAAAGCCAUGAAGCGCUACGCCCUGGUGCCCGGCACCAU
CGCCUUUACCGACGCACAUAUCGAGGUGGACAUUCCUAC
GCCGAGUACUUCGAGAUGAGCGUUCGGCUGGCAGAAGCUA
UGAAGCGCUAUUGGGCUGAAUACAAACCAUCGGAUCGUGGU
GUGCAGCGAGAAUAGCUUGCAGUUCUCAUGCCCGUGUUG
GGUGCCCUGUUCAUCCGGGUGUGGCUGUGGCCCCAGCUAACG
ACAUCUAACGAGCGGCGGAGCUGCUGAACAGCAUGGGCAU
CAGCCAGGCCCACCGUCGGUAUUCGUGAGGAAGAAGGGCUG
CAAAAGAUCCUCAACGUGCAAAAAGAAGCUACCGAUCAUAC
AAAAGAUCAUCAUCAUGGAUAGGCAAGACCGACUACCAGGG
CUUCCAAAAGCAUGUACACCUCGUGACUUCCUUCCAUUGCCA
CCCGGCUUCAGAUACGACUUCGUGCCCGAGAGCUUCG
ACCGGGACAAAACCAUCGCCCUGAUCAUGAACAGUAGUGG
CAGUACCGGAGAUUGCCCAAGGGCGGUAGCCCUACCCGCACCGC
ACCCGCUUGUGUCCGAUUCAUCAUGCCCGCCGACCCAUCU
UCGGCAACCAGAUCAUCCCCGACACCGCCUAUCCUCACCGU
GGUGCCAUUCACCACCGCGCUUCGGCAUGUUCACCACCUGCUG
GGCUACUUGAUCUGCGGCUUCUGGUCGGUGCUCAUGUACC
GCUUCGAGGGAGGAGCUAUUCUUGCGCAGCUUGCAAGACUA
UAAGAUUCAAUCUCCCCUGCUGGUGCCCACUAUUAGC
UUCUCCGCUAAAGAGCACUCUCAUCGACAAGUACGACCUAA
GCAACUUGCACGAGAUCGCCAGCGCGCGGGCGCCGCUCAG
CAAGGAGGUGUGGUGAGGCCGUGGCCAAACGGCUUCACCUA
CCAGGCAUCCCGCCAGGGCUACGGCCUGACAGAAACAAACCA
GCGCCAUCUGAUCCCCCCCGAAGGGGACGACAAGCCUGG
CGCAGUAGGCAAGGUGGUGCCCCUUCUCUGCGCUAAAGGUG
GUGGACUUGGACACCGGUAAGACACUGGGGUGAACCAGC
GCGGGCGAGGCUGUGCGUCCGUGGCCCCAUGAUCAUGAGCGG
CUACGUUAACAACCCCGAGGCUACAAACCGCUCUCAUCGAC
AAGGACGCGCUGGCUGCACAGCGGCGACAUCCGCCUACUGGG
ACGAGGACGAGCACUUCUCUCUGUGGACCGGCUGAAGAG
CCUGAUCAAAUACAAAGGGCUACCAGGUAGCCCCAGCCGAA
CUGGAGAGCAUCCUGCUGCAACACCCCCAACAUUCGACG
CCGGGGUGUCCGCCGGCCUGCCCCGACGACGAUGCCGGCGAGCU
GCCCGCCCGCAGUCGUCGUGCUGGAACACGGGUAAAAACCAUG
ACCGAGAAGGAGAUGUGUGGACUAUGUGGCCAGCCAGGUA
CAACCGCCAAGAAGCUGCGGCGGUGGUGUGUGUGUGUGGA
CGAGGUGCCUAAAGGACUGACCGGCAAGUGGACGCCCGC
AAGAUCCGCGAGAUUCUCAUUAAGCCAAGAAGGGCGGCA
AGAUCGCCGUGUAAY
Human Factor IX (FIX) mRNA:
XAUGCAGCGCGGUGAACAUGAUCAUGGCAGAAUCACCAGGC
CUCAUCACCAUCUGCCUUUUAGGAUAUCUACUCAGUGCUG
AAUGUACAGUUUUCUCAUGAUCAUGAAAACGCCAACAAAAAU
UCUGAGGCGGAGAAGGAGGUAUAAUUCAGGUAAAAUGGAA
GAGUUUGUUCAAGGGAACCUUGAGGAGAAUGUAUGGAAG
AAAAGUGUAGUUUUGAAGAAGCACGAGAAGUUUUGAAAA
CACUGAAAGAACAACUGAAUUUUGGAAGCAGUAUGUUGAU
GGAGAUCAGUGUGAGUCCAAUCCAUGUUUAAAAUGGCGCGCA
GUUGCAAGGAUGACUUAAUUCCUAUGAAUGUUGGUGUCC
CUUGGAUUGAAGGAAAGAACUGUGAAUUAGAUGUAACA
UGUAACAUUAGAAUGGCAGAUGCGAGCAGUUUUGUAAAA
AUAGUGCUGAUAACAAAGGUGGGUUGCUCCUGUACUGAGGG
AUAUCGACUUGCAGAAAACCAGAAGUCCUGUGAACCAGCA
GUGCCAUUCCAUGUGGAAGAGUUCUGUUUCAAACUU
CUAAGCUCACCGUGCUGAGGCGCUGUUUUUCCUGAUGUGGA
CUAUGUAAAUCUACUGAAGCUGAAAACCAUUUGGAUAAC
AUCACUCAAAAGCACCCAAUCAUUUAAUGACUUCACUCUGGG
UUGUGUGUGGAGAAGAGACCAAAACCAGGUCAAUUCCUUG
GCAGGUUGUUUGAAUGGGUAAAGUGAUGAUGCAUUCUGUGGA
GGCUCUAUCGUUAAUGAAAAAAUGGAUUGUAACUGCUGCCC
ACUGUGUGAAAACUGGUGUUAAAAAAUACAGUGUCAGCG
UGAACAUAAAUAUUGAGGAGACAGAACAUACAGAGCAAAAG
CGAAAUGUGAUUCGAAUUAUUCCUCACCACACUACAAUG
CAGCUAUUAAUAAAGUAACCAUGACAUGCCCUUCUGGA
ACUGGACGAACCCUUAGUGCUAAACAGCUACGUUACCU
AUUGCAUGCUGACAAGGAAUACACGAACAUCUUCUCUCA
AAUUGGAUCUGCGCUAUGUAAAGUGCGCUGGGGGAAGAGUCUCUU
CCACAAAGGGAGAGAUCAGGCUUUAGUUCUCAUACCUUAGA
GUUCCACUUGUGUGACGAGCCACAUGUCUUCGAUCUAAA
AGUUCACCAUCUAUAAACAAACAUGUUCUGCUGGCUUCCA
UGAAGGAGGUGUAGAGAUUCAUGUCAAGGAGAUAGUGGGGGA
CCCCAUGUUACUGAAGUGGAAGGGACCAGUUCUGUAACUG
GAAUUAUAGCUGGGGGUGAAGAGUGUGCAAUGAAAGGCAA
AUAUGGAAUAUAUACCAAGGUAUCCCGGUAUGUCAACUGG
AUUAAGGAAAAAAAACAAAGCUCACUUAY
Codon-optimized human phenylalanine hydroxylase (PAH) mRNA:
XAUGAGCACCGCCGUGCUGGAGAACCCCGGCCUGCGCCGC
AAGCUGAGCGACUUCUGCGCCAGGAGACCAGCUACAUCGAGG
ACAACUGCAACCAGAACGGCGCGCCAUCAGCCUGAUCUCAG
CCUGAAGGAGGGAGGUGGGCGCCCGCGCCAAAGGUGCUGCGCC
CUGUCGAGAGAGAACGACGUGAACCUGACCACAUCGAGA
GCCGCCCCAGCCGCCUGAAGAAGGAGCGAGUACGAGUUCUU
CACCCACCUGGACAAGCGCAGCCUGCCCGCCCUGACCAAC
AUCAUCAAGAUCCUGCCCGCCACGACAUCGGCGCCACCGUGC
ACGAGCUGAGCCGGCGACAAGAAGAAGGACACCGUGCCCUG
GUUCCCCCGCACCAUCCAGGAGCUGGACCGCUUCGCCAAC
CAGAUCCUGAGCUACGCGCGCCGAGCUGGACGCCGACCACC
CCGGCUUCAAGGACCCCGUGUACCCGCGCCCGCCGCAAGCA
GUUCGCCGACAUCCGCCUACACUACCGCCACGGCCAGCCC
AUCCCCCGGCGUGGAGUACAUGGAGAGGAGAGAAGACCU
GGGGCACCGUGUUCAAGACCCUGAAGAGCCUGUAGADAC
CCACGCCUGCUACGAGUACAACCACAUCUUCCCCCUGCUG
GAGAAGUACUGCGGCUUCCCACGAGACAACAUCCCCAGC
UGGAGGACGUGAGCCAGUUCCUGCAGACCUGCACCGGCUU
CCGCCUGCCGCCCCGUGGCCGGCCUGCUGAGCAGCCGGAC
UUCCUGGGGCGCCUGGCCUUCCGCGGUGUUCCACUGCACCC
AGUACAUCCCGCCACGGCAGCAAGCCCAUGUAACCCCCCGA
GCCCGACAUCUGCCACGAGCUGCUGGCGCCACGUGCCCCUG
UUCAGGCGACCGCAGCUUCCGCCCAGUUCAGCCAGGAGAUCG
GCCUGGCCAGCCUGGGCGCCCCCCGACGAGUACAUCGAGAA
GCUGGCCACCAUCUACUGGUUCACCGUGGGAGUUCGGCCUG
UGCAAGCAGGCGCGACAGCAUGCAAGGCCUACGGCGCCGGCC
UGCUGAGCAGCUUCGUCGGCGAGCUGCAGUACUGCCUGACGA
GAAGCCCAAGCUGCUGCCCCUGGAGCUGGAGAAGACCGCC
AUCCAGAACUACACCGUGACCGAGUUCCALCCCUGUACU
ACGUGGCCGAGAGGCUUCAACGGCCAAAGGAGAAGGUGCG
CAACUUCCGCCGCACCAUCCAUCCCCCCGCCCCUCUCCGGUGCGC
UACGACCCCUAACCCCAGCGCAUCGAGGUGCUGGACAACA
CCCAGCAGCUGAAGAUCCUGGCCGACAGCAUCAAGCGA
GAUCGGCAUCCUGUGCAGGCGCCCUGCAGAAGAUCAAGUAA
Y
Codon-optimized cystic fibrosis transmembrane conductance regulator (CFTR) mRNA:
AUGCAGGCGGUCCCCGCUCGAAAAGGCCAGUCUGUCUCCA
AACUCUUCUCUCAUGGGACUCGGCCUAUCCUUAGAAAGGG
GUAUCGGGCAGAGGCUUGAGUUGUCUCAUCUACCAGAUC
CCCUCGGUAGAUUCGGCGGAUAACCUCUCCGGAGAAGCUCG
AACGGGAAUGGGACCGCGAACUCGCGUCUAAGAAAAACCC
GAAGCUCAUCAGCACUGAGAAGGUGCUUCUCUCUGCGCG
UUCAUGUUCUGGUAUCUUCUCUGUAUCUCGGGGGAGGUCA
CAAAAGCAGUCCAACCCCUGUGUGUGUGGUCGCAUUAUCGC
CUCGUACGACCCCGAUAACAAAAGAAGAGGAGCAUCGCG
AUCUCCUCGGAUCGGGACUGUGUUUGCUUUUCAUCAUCGUCA
GAACACUUUUGUCAUCCAGCAAUCUUCUGCCUCAUCA
CAUCGGUAUGCAGAUGCGAAUCGCUAUGUUUAGCUUGAUC
UACAAAAAGACACUGAAACUCUCGUCGCGCGGUGUUGGAUA
AGAUUCCAUCGGGUCAGUUGGUGUCCCUGCUUAGUAAUAA
CCUCAACAAAUUCGAUGAGGGGACUGGCGCUGGCACAUUC
GUGUGGAUUGCCCCGUUGCAAGUCCGCCCUUUGGGCC
UUAUUUGGGGAGCUGUUGCAGGCAUCUGCCUUUGUGGCCU
GGGAUUUCUGAUUGUGUGUGGCAUUGUUUCAGGGCUGGGGCUU
GGGCGGAUGAUGAUGAUGAGUAUCGCGACCAGAGAGCGGUA
AAAUCUGGAAAGACUCCGUCAUCAUCCGAAAUGAUCGA
AAACAUCCAGUCGGUCAAAAGCCUAUGCUGGGAAGAAGCU
AUGGAGAAGAUGAUGAAAACCUCCGCCAAACUGAGCUGA
AACUGACCCGCAAGGCGGCGGUAUGUCCGGGUAUUUCAAUC
GUCAGCCGUUCUUCUUCCGGGGUUCUCUGUGUGUCUCUCUCUC
UCGGUUUGCCUUAUGCCUUGAUUAAGGGGAUUAUCCUCC
GCAAGAUUUUCACCACGAUUCGUUCUGCAUGAUUGUGCG
CAUGGCAGUGACACGGCAAUUUCCGGUGGGGCCGUGCAGACA
UGGUAUGACUCGCUUGGAGCGAUCAAAAAUCCAAGACU
UCUUGCAAAAGCAAGAGUAGAAGACCCCUGGAGUACAAUCU
UACUACUACGGAGGGUAGUAAUGGGAAUGUGAGCGCUUUU
UGGGAAGAGGGGUUUGGAGAACUGUUGAAAGCAAAGC
AGAAUAACAACAACCGCAAGACCUCAAAUGGGAGACGAUC
CCUGUUUUCUCGAACUUCUCCUGCCUCGGAACACCCGUG
UUGAAGGACAUCAAUUCAAGAUUGAGAGGGACAGCUUC
UCGCGGUAGCGGAAGCACUGGUGCGCGGAAAAAACUAGCCU
CUUGAUGGUGAUUAUGGGGGGAGGCUUGACCCCAGGCGGGGG
AAGAUUAAAACUCUCCGGGCGGUAUCUCUCUCUGUGUAGCCAGU
UUUCAUGGAGAUCAUGCCCGGAACCAUUAAAGAGAACAUCAU
UUUCGGAGUAUCCUAUGAUGAGUACCGAUACAGAUCGGUC
AUUAAGGCGUGCCAGUUGGAAGAGGACAUUUCUAAGUUCG
CCGAGAAGGAUAACAUCGUCUUGGGAGAAGGGGUAUUC
AUUGUCGGGAGGGGCAGCGAGGCGCGGAUCAGCCUCGCGAGA
GCGGUAUAAAAGAUGCAGAUUUGUAUCUGCUUGAUCAC
CGUUGGAUACCUCGACGUAUGACAGAAAAAGAAAUCUU
CGAGUCGUGCGGUGUAAAACUUAUGGCUAAUAAGACGAGA
AUCCUGGUGAGACAUCAAAAAAGGAACACCUAAGAAGGCGG
ACAAGAUCCUGAUCCUCCACGAAGGAUCGUCCUACUUUA
CGGCACUUCUCAGAGUUGCAAAACUUGCAGCCGGACUUC
UCAAGCAAACUCAUGGGGUGUGACUCAUCACGACUUCA
GCCGGAACGGCGGAACUCGAUCUGACGGGAAAACCUGCA
CCGAUUCUCCGCUUGAGGGGUGAUGCCCCGGUAUCGUGGACC
GAGACAAAGAAGCAGUUCGUUAAGCAGACAGGAGAAUUG
GUGAGAAAAAGAAAGAACAGUAUCUUGAUCCUAUUAACUC
AAUUCGCAAGUUCUCAUCAAUCCGUCCAGAAAACUCCACUGCAG
AUGAAUGGAAUUGAAGAGGAUCGGACGAACCCCUGGAGC
GCAGGCUAGCCUCGUGCCGGAUCAGAGGCAAGGGGAGGC
CAUUCUCCCCGGAUUCGGGUGAUUCACCGGACCUACA
CUUCAGGCGAGGGCGAAGGCAAUCCGUGCUCAACCUCAUGA
CGCAUCUGGUAAACCAGGGGGCAAAACAUCACCGCAAAAC
GACGGCCUCAACGAGAAAAGUGUCACUUGCACCCCAGCGG
AAUUGACUGAACUCGACAUCUCAGCCGUAGGCUUCGC
AAGAAACCGGACUUGAGAUCGAGCGAAGAAAUCAAUGAAGA
AGAUUGAAAGAGUUGUUCUUGAUGACAUGGAAUCAAUC
CCAGCGGUGACAACGUGGAACACAUACUUGCGUUACAUCA
CGGUGCACAAGUCCUUGAUUGUUCGUCCUCUCUCUGGUGUCU
CGUGAUCUUCUCCGCUGAGGGUCGCAGCGUCACUUGUGGUC
CUCUGGCUGCUUGGUAAAUACGCCCCUUGCAAGACAAAGGCA
AUUCUACACUCAAGAAACAAAUUCCUAUGCCUGUGAUUAU
CACUUCUAGAGCGUAUUAUCGUGUUUUACUACGUA
GGAGUGGCCGACACUCUGCUCGCGAUGGGGUUUCUCCGAG
GACUCCCACUCGUUCACAGCUUAUCUCUCACAGAU
UCUCCACCAUAAGAUGCUUCAUAGCCGUACUGCAGGCUCCC
AUGUCCACCUGAAUACCGCUCAAGGCGGGAGGUGUAUUUGA
AUCGCUUCAUCAAAAGAUAUUGCAAUUUUGGAUGACCUUCU
GCCCCUGACGAUCGUUCGACUUCAUCCAGUGUGCUGAUC
GUGAUUGGGCGCUAUUGCAGUAGUCGUGUCCUCCUCCAGCCUU
ACAUUUUUGUCGCGACCGUUCCGGUGAUCGUGGGCGUUUAU
CAUGCUGCGGGCCUAUUUCUGCAGACGUCACAGCAGCUU
AAGCAACUGGAGUCUGAAGGGGAGGGUCGCCUAUCUCUCGC
AUCUUGUGACCAGUUGAAGGGAGAUGUGGAGACGUUGCGCGC
CUUGGCAGGCAGCCCCUACUUGAAACACUGUUCCAAAA
GCGCUGAAUCUCCAUACGGCAAAAUUGGUUUUUGUAUUUGA
GUACCCUCCGAUGGUUUCAGAUGCGCAUGAGUGAUUUU
UGUGAUCUCUCHUUAUCCGCGGUGACUUUUAUCUCAUCUUG
ACCACGGGAGAGGGGGCGAGGGACGGGGUCGGGUAUUAUCCUGA
CACUCGCCAUGAACAUUAUGAGCACUUGCAGUGGGGCAGU
GAACAGCUCGAUGAUGAUGUGGAUAGCCUGAUGAGGUCCGUU
UCGAGGGUUCUUAAGUUCAUCCGACAUGCCGACGGAGGGAA
AGCCCACAAAAAAGUAACGAAAACCCUAUAAGAAUGGGCAAUU
GAGUAAGGUAAAUGAUCAUCGAGAACAGUCACGUGAAGAAG
GAUGACAUCUGCCGUAGCGGGGGGUCAGAUGACGUGAAGG
ACCUGACGGCAAAAAAUACCGAGGGAGGGAACGCAAUCCU
UGAAAACAUCUCGUUCAGUAGCCCCCGGUCAGGCGUGUG
GGGUUGCUCGGGGAGGACCGGGUGUCAGAAAAAUCCGACGUGCC
UGUCGGCCUCUCUGUGAGAUCUUCUGAAAUACAGAGGGUGAGAU
CCAGAUCGACGGCGUUUCGUGGGAUAGCAUCACCUGCAG
CAGUGGCGGAAAAGCGGUUGGAGUAAAUCCCCCAAAAGGUCU
UUAUCUUAGACGGAACCUCUCGAAAGAAUCUCGAUCCUA
UGAACAGUGGUCAGAUAGAGAGAUUGGAAAGUCGCGGAC
GAGGUUGGCCUUCGGGAGUGUAAUCGAGGAUUCCGGGAA
AACUCGACUUUGUCCUUGUAGAUGGGGGGAUGCCGUCCUGUC
GCAUGGGCACAAGCAGCUCAUGUGCCUGGCGCGAUCCGUC
CUCUCUAAGCCGAAAAUCUUCUCUCUGGAUGAACCUCUGG
CCCAUCUGGACCCGGUAAACGUAUCAGAUCAUCAGAAGGAC
ACUUAAGCAGGCGGUUGCCGAAGUGCACGGGUGAUUCUCUGU
GAGCAUCGUAUCCGAGGCCAUGCUCGAAUGCCAAGCAAUUC
UUGUCAUCCGAAGAGAAUAAAGGUCCGCCAGUACGACUCCAU
CCAGAAGCUGCUUAAUGAGAGAGAUCAUGUUCCGCGCAGGCG
AUUUCACCAUCCGAUAGGGUGAAACUUUUCCACAGAA
AUUCGUCGAAGUGCAAGUCCAAAACCGCAGAUCCGCGGCCUU
GAAAGAAGAGACUGAAGAAGAAGUUCAAGACACGCGCUCUU
UAA
5'and 3'UTR array X =
GGACAGAUCCGCCUGGAGACGCCAUCCACCGCUGUUUGACC
UCCAUAGAAGACACCGGGACCGAUCCAGCUCCGCGCGCG
GGAACGGUGCAUUGGAACGGCGAUCCCCGUGCCAAGAU
GACUCACCGUCCUUGACAG
Y =
CGGGUGGCAUCCUCCCUGUGACCCCUCCCAGUGCCUCUCCUG
GCCCUGGAAGUGCCACUCCAGUGCCCACCAGCCUGUCC
UAAUAAAAAUUAAGUGCAUCAAGCU

Lipid Nanoparticle Preparation An ethanolic solution of a mixture of lipids (cationic lipids, helper lipids, twin ion lipids, PEG lipids, etc.) was prepared to the reported volume and heated to the temperature of choice. Individually, a buffered solution of mRNA (10 mM citrate / 150 mM NaCl, pH 4.5) was prepared from a 1 mg / mL stock solution and heated to the selected temperature for 5-10 minutes.

For small-scale preparations, the lipid solution is rapidly injected into the mRNA aqueous solution using a syringe pump (3.71 mL / sec) and the resulting suspension is shaken to contain the lipid nanoparticles 20. % Ethanol was obtained. The resulting nanoparticle suspension was diafiltered with 1 x PBS (pH 7.4), concentrated and stored at 2-8 ° C.

Representative Examples at 25 ° C. A certain amount of a 50 mg / mL ethanolic solution of cKK-E12, DOPE, Chol and DMG-PEG2K was mixed and diluted with ethanol to obtain a final volume of 3 mL. Individually, a buffered aqueous solution of FFL mRNA (10 mM citrate / 150 mM NaCl, pH 4)
.. 5) was prepared from a 1 mg / mL stock solution. The lipid solution was rapidly injected into the mRNA aqueous solution and shaken to give a final suspension of 20% ethanol. The resulting nanoparticle suspension was filtered, diafiltered with 1 × PBS (pH 7.4), concentrated and stored at 2-8 ° C. Final concentration = 0.20 mg / mL FFL mRNA (encapsulated). Z _ave =
91 nm PDI (0.16).

Preparation at 37 ° C. A certain amount of a 50 mg / mL ethanolic solution of cKK-E12, DOPE, Chol and DMG-PEG2K was mixed and diluted with ethanol to obtain a final volume of 3 mL. Individually, a buffered aqueous solution of FIX mRNA (10 mM citrate / 150 mM NaCl, pH 4)
.. 5) was prepared from a 1 mg / mL stock solution. The lipid solution was rapidly injected into the mRNA aqueous solution and shaken to give a final suspension of 20% ethanol. The resulting nanoparticle suspension was filtered, diafiltered with 1 × PBS (pH 7.4), concentrated and stored at 2-8 ° C. Final concentration = 0.20 mg / mL FIX mRNA (encapsulation). Z _ave = 6
4 nm; PDI (0.12).

Preparation at 65 ° C. A certain amount of a 50 mg / mL ethanolic solution of cKK-E12, DOPE, Chol and DMG-PEG2K was mixed and diluted with ethanol to obtain a final volume of 3 mL. Individually, a buffered aqueous solution of FIX mRNA (10 mM citrate / 150 mM NaCl, pH 4)
.. 5) was prepared from a 1 mg / mL stock solution. The lipid solution was rapidly injected into the mRNA aqueous solution and shaken to give a final suspension of 20% ethanol. The resulting nanoparticle suspension was filtered, diafiltered with 1 × PBS (pH 7.4), concentrated and stored at 2-8 ° C. Final concentration = 0.20 mg / mL FIX mRNA (encapsulation). Z _ave = 7
3 nm; PDI (0.13).

Effect of temperature on nanoparticle encapsulation process Ethanol lipid solution and buffered aqueous solution of mRNA (10 mM citrate / 150 mM N)
Both aCl, pH 4.5) were heated at different selective temperatures prior to the formulation process to determine the effect of temperature on the final yield and encapsulation efficiency of the formulation.

The effect of temperature on the nanoparticle formulation process was evaluated for size, size dispersibility, encapsulation efficiency and yield (or recovery). Illustrative data are shown in Table 1. As can be seen, an increase in temperature (eg, above ambient temperature) results in an increase in encapsulation efficiency and / or yield / recovery, as well as a decrease in particle size and / or size dispersibility.

Example 2. Enlarged-Scale Formulation Process This example illustrates an exemplary scale-up formulation process for encapsulating mRNA at increased temperature.

An exemplary scaled formulation process is shown in FIG. An Ismatec programmable digital drive pump (Cole Palmer model number CP7808-10) was used. Micropump A-mount suction shoe pump head 316 SS body / graphite gear / PTFE seal, 0.084mL / rev, no internal bypass (Cole)
Palmer model number 07002-27) and Pharma Pure tube size #
14, 0.06 "ID, 1/16" (Spectrum labs part number ACTU-P14
-25N) was used.

Nanoparticle preparations and mRNA encapsulation are performed by "T" the ethanol lipid solution and mRNA in citrate buffer (10 mM citrate buffer, 150 mM NaCl, pH 4.5).
Prepared by mixing using a junction (or "Y" junction). An exemplary flow rate of a citrate buffer containing mRNA and an ethanol solution containing lipids is
200 ml / min and 50 ml / min, respectively. During this process, both pumps are started at the same time. Fractions at the start and end of the product were discarded and only the intermediate product was collected. Accurate flow velocity and low pulse flow are two important parameters of this process.

Purification and buffer exchange Purification and buffer exchange of the pharmaceutical product obtained from the above steps is carried out in a KrosFlo® Research IIi tangential flow filtration system from Spectram labs using a modified polyether sulfone hollow fiber filter module. .. Buffer exchange is performed in 6x volume sterile PBS (pH 7.4) in continuous diafiltration form. See FIG. The pharmaceutical products are analyzed for size (PDI) and encapsulation (yield). Illustrative data are shown in Table 2.

Using this process, a very narrow particle size range and high encapsulation efficiency (eg, an average of over 90%) are achieved.

To test the importance of low pulse homogeneous flow, a peristaltic pump with some pulsatile flow was used for the pharmaceutical process. See FIG. Citrate buffer containing mRNA and pure ethanol containing lipid were mixed at flow rates of 200 ml / min and 50 ml / min, respectively. Illustrative results are shown in Table 3. As can be seen, the use of peristaltic pumps within this process results in a formulation of nanoparticles with a larger size. This is likely due to a heterogeneous mixture due to pulsatile flow.

Equivalents and Scope One of ordinary skill in the art will recognize many equivalents for a particular embodiment of the invention described herein, or will be able to confirm using only routine experiments. The scope of the present invention is not intended to be limited by the above description, but is specified in the following claims.

Claims (13)

A nanoparticle formulation containing lipid nanoparticles that encapsulate mRNA encoding a cystic fibrosis conductance control protein.
Here, the mRNA encoding the cystic fibrosis conductance control protein has a length of more than 4 kb and is encapsulated within the lipid nanoparticles with an encapsulation efficiency of 85% or more.
Here, the lipid nanoparticles have an average size of 80 nm or less and a homogeneity scale (PDI) of 0.19 or less.
Nanoparticle formulation. The nanoparticle preparation according to claim 1, wherein the lipid nanoparticles include one or more cationic lipids, one or more helper lipids, and one or more PEG lipids. The one or more cationic lipids are C12-200, MC3, DLinDMA, DLinkC2DMA, cKK-E12, ICE (imidazole type), HGT5000, HGT5001, DDAB, DOSPA, DOGS, DODAC, DODA, DMDMA, DODAC, DLendam. The nanoparticle preparation according to claim 2, which is selected from the group consisting of DMRIE, CLINDMA, CpLinDMA, DMOBA, DOcarbDAP, DLinDAP, DLincarbDAP, DLinCDAP, Klin-K-DMA, DLin-K-XTC2-DMA, and HGT4003. .. The one or more helper lipids are DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE (1, 2-Giorail-sn-glycero-3-phosphoethanolamine), DOPC (1,2-diorail-sn-glycero-3-phothhotidylcholine), DPPE (1,2-dipalmitoyl-sn-glycero-3-3) Phosphoethanolamine), DMPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), and DOPG (1,2-dioleoil-sn-glycero-3-phospho- (1'-rac-glycerol)) The nanoparticle preparation according to claim 2, which is selected from. The nanoparticle preparation according to claim 2, wherein the lipid nanoparticles further contain one or more cholesterol-based lipids. The nanoparticle preparation according to claim 1, wherein the lipid nanoparticles have a PDI of 0.12 or less. The nanoparticle preparation according to claim 1, wherein the lipid nanoparticles have a PDI of 0.10 or less. The nanoparticle preparation according to claim 1, wherein the lipid nanoparticles have an encapsulation efficiency of more than 90%. The nanoparticle preparation according to claim 1, wherein the lipid nanoparticles have an encapsulation efficiency of more than 95%. The nanoparticle preparation according to claim 1, wherein the mRNA-loaded lipid nanoparticles have a lipid / mRNA (N / P) N / P ratio greater than 1. The nanoparticle preparation according to claim 10, wherein the mRNA-loaded lipid nanoparticles have a lipid / mRNA (N / P) N / P ratio of about 2 to about 6. The nanoparticle preparation according to claim 1, wherein the lipid nanoparticles contain a PEG-modified lipid. The nanoparticle preparation according to claim 12, wherein at least 5% of the total lipids in the lipid nanoparticles are PEG-modified lipids.

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